running head: antiviral responses in the model monocot ... · 06.09.2012 · pmv include foxtail...

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Running Head: Antiviral responses in the model monocot Brachypodium

Corresponding Author: Karen-Beth G. Scholthof, Department of Plant Pathology and

Microbiology, Texas A&M University, 2132 TAMU, College Station, TX, USA

Telephone: +1-979-845-8265

E-mail: [email protected]

Research Category: Plants Interacting with Other Organisms

Plant Physiology Preview. Published on September 6, 2012, as DOI:10.1104/pp.112.204362

Copyright 2012 by the American Society of Plant Biologists

https://plantphysiol.orgDownloaded on March 28, 2021. - Published by Copyright (c) 2020 American Society of Plant Biologists. All rights reserved.

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Characterization of a viral synergism in the monocot Brachypodium reveals distinctly

altered host molecular processes associated with disease1

Kranthi K. Mandadi and Karen-Beth G. Scholthof *

Department of Plant Pathology and Microbiology (K.K.M., and K-B.G.S.), Texas A&M

University, 2132 TAMU, College Station, TX 77843



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1This work was funded by THECB-ARP (000517-0002-2009) awarded to K-B.G.S.

*Corresponding author; email [email protected]

Fax: +1-979-845-6483

The author responsible for distribution of materials integral to the findings presented in this

article in accordance with the policy described in the Instructions for Authors

(www.plantphysiol.org) is: Karen-Beth G. Scholthof ([email protected]).



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ABSTRACT

Panicum mosaic virus (PMV) and its satellite virus (SPMV) together infect several small grain

crops, biofuel, forage and turf grasses. Here, we establish the emerging monocot model

Brachypodium distachyon as an alternate host to study PMV- and SPMV-host interactions and

viral synergism. Infection of Brachypodium with PMV+SPMV induced chlorosis and necrosis of

leaves, reduced seed-set, caused stunting, and lowered biomass, more than PMV alone. Towards

gaining a molecular understanding of PMV- and SPMV-affected host processes, we used a

custom-designed microarray and analyzed global changes in gene expression of PMV and

PMV+SPMV infected plants. PMV infection by itself modulated expression of putative genes

functioning in carbon metabolism, photosynthesis, metabolite transport, protein modification,

cell-wall remodeling and cell-death. Many of these genes were additively altered in a co-

infection with PMV+SPMV and correlated to the exacerbated symptoms of PMV+SPMV co-

infected plants. PMV+SPMV co-infection also uniquely altered expression of certain genes

including transcription and splicing-factors. Among the host defenses commonly affected in

PMV and PMV+SPMV co-infections, expression of an antiviral RNA-silencing component,

SILENCING DEFECTIVE 3 (SDE3) was suppressed. Several salicylic acid (SA) signaling

components such as PR genes and WRKY transcription factors were up-regulated. In contrast,

several genes in jasmonic acid (JA) and ethylene (ET) responses were down-regulated.

Strikingly, numerous protein kinases including several classes of receptor-like kinases were mis-

expressed. Taken together, our results identified distinctly altered immune responses in monocot

antiviral defenses, and provide novel insights into monocot viral synergism.



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INTRODUCTION

In the past decades substantial progress has been made in dissecting interactions between

plant and virus proteins and to define the role of virus-encoded proteins in plant cells. Model

plant species such as Arabidopsis thaliana (Arabidopsis) and Nicotiana benthamiana have been

crucial in determining the mechanics of antiviral defenses. Genome-wide expression studies in

these model plants identified networks of genes affected commonly by several plant viruses,

whereby diverse DNA and RNA viruses alter genes in primary and secondary metabolism,

detoxification, cell-wall remodeling, and defense responses (Whitham et al., 2003; Whitham et

al., 2006; Ascencio-Ibáñez et al., 2008; García-Marcos et al., 2009; Elena et al., 2011; Hanssen

et al., 2011; Postnikova and Nemchinov, 2012). At the cellular level, specific recognition of viral

nucleic acids or viral proteins by host resistance gene (R-gene) products inhibits viral replication

or accumulation (Kohm et al., 1993; Bendahmane et al., 1999; Ishibashi et al., 2007).

Additionally, tissue-level hypersensitive response (HR) and systemic acquired resistance (SAR),

often mediated by defense hormones such as salicylic acid (SA), both delimit movement of the

virus from the primary infected cells, and induce a broad spectrum resistance to systemic

infection (Huang et al., 2005; Alamillo et al., 2006; Kachroo et al., 2006; Heil and Ton, 2008;

Baebler et al., 2011). Recently, RNA silencing has emerged as a broadly conserved defense

response against viruses (Dunoyer and Voinnet, 2005; Alvarado and Scholthof, 2009; Alvarado

and Scholthof, 2012). Multiple RNA silencing components such as Dicer, double-stranded RNA-

binding (DRB) protein, HUA enhancer 1 (HEN1), Argonaute (AGO), and RNA-dependent RNA

polymerase (RDR) play critical roles in programming virus-specific RNA-induced silencing

complexes (vRISC) that subsequently target viral RNA for degradation (Alvarado and Scholthof,

2009). As a co-evolutionary strategy, generally referred to as the Red Queen Hypothesis (Van

Valen, 1973), to antagonize host antiviral defenses, many viruses encode suppressor proteins that

function to interfere and suppress RNA silencing (Dunoyer and Voinnet, 2005; Alvarado and

Scholthof, 2012). These and other host:virus outcomes have been recently summarized by Pallas

and Garcia (Pallas and García, 2011).

Interestingly, viral suppressors were discovered by studying a mixed virus infection that

induced a synergism on tobacco plants. This co-infection of Potato virus X (PVX, genus

Potexvirus) and Potato virus Y (PVY, genus Potyvirus) on potato and tobacco plants resulted in

significantly increased levels of PVX compared to single virus infections, and greatly enhanced



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symptoms (Rochow and Ross, 1955; Damirdagh and Ross, 1967; Goodman and Ross, 1974;

Vance, 1991). PVY encodes a potent silencing suppressor protein, HC-Pro, which enhances

accumulation of PVX in tobacco (Pruss et al., 1997; González-Jara et al., 2004; Pruss et al.,

2004), but not in N. benthamiana suggesting that viral synergistic interactions can be host-

specific (García-Marcos et al., 2009).

Much of our knowledge of virus:host interactions has been determined under laboratory

conditions with dicotyledonous host plants, especially N. benthamiana and Arabidopsis. Studies

of viruses with host ranges limited to monocots (plants in the Poaceae, including forage and turf

grasses and small grains) have lagged due to the lack of a tractable model species. Similarly, few

studies of mixed virus infections have been reported on monocot or dicot host plants, resulting in

a gap in our molecular understanding of how plants respond to mixed (or synergistic) virus

infections. For example, little is known about how viral synergism alters host defenses and

whether antiviral defenses are conserved amongst dicot and monocot plants. Here, we have

focused on addressing the biological response of Brachypodium distachyon (Brachypodium), an

emerging model grass species, to the mixed infection of Panicum mosaic virus (PMV) and its

satellite virus (SPMV).

PMV, the type member of the genus Panicovirus in the family Tombusviridae, is a single-

stranded, positive-sense RNA with a host range limited to the Poaceae family of grasses (Turina

et al., 1998; Scholthof, 1999; Turina et al., 2000). PMV was first described in natural infections

of switchgrass (Panicum virgatum) (Sill and Pickett, 1957) and subsequently as the causal agent

of “St. Augustine decline” disease on St. Augustinegrass (Stenotaphrum secundatum), a popular

turfgrass in the southern United States (Cabrera and Scholthof, 1999). Experimental hosts of

PMV include foxtail millet (Setaria italica), pearl millet (Pennisetum glaucum), and proso millet

(Panicum miliaceum) (Buzen et al., 1984). From its 4,326 nucleotide (nt) genomic RNA (gRNA)

PMV expresses two replicase-associated proteins (P48 and P112), using translation read-through

of the 48-kDa open reading frame to express the 112-kDa protein (Batten et al., 2006b) (Figure

S1). Four genes are encoded on the subgenomic RNA (sgRNA) that facilitate virus movement

(P8, P6.6, P15) and encapsidate the gRNA (26-kDa capsid protein, CP) to form 30 nm (T=3)

virions (Turina et al., 2000; Batten et al., 2006b).

PMV also supports the replication and systemic spread of satellite panicum mosaic virus

(SPMV), an 824 nt positive-sense single-stranded RNA (Niblett and Paulsen, 1975; Masuta et



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al., 1987; Turina et al., 1998; Scholthof, 1999) (Figure S1). SPMV gRNA encodes a 17-kDa CP

(SPMV CP) to form 16 nm (T=1) icosahedral particles (Ban et al., 1995; Desvoyes and

Scholthof, 2000). Previously, we have shown that in addition to encapsidation, SPMV CP

localizes to the host plasma membrane forming puncta near the plasmodesmata (Qi et al., 2008),

reflecting its movement-protein like functions. SPMV CP also traffics to the nucleus and

nucleolus (Qi et al., 2008) forming Cajal body-like structures similar to other nucleolar-localized

viral proteins (Kim et al., 2007). Additionally, SPMV CP induces a non-host hypersensitive

response in N. benthamiana when expressed from viral gene vectors (Qiu and Scholthof, 2004),

prevents the accumulation of defective interfering SPMV RNA in PMV mixed infections of

millets (Qiu and Scholthof, 2001), and stabilizes foreign gene inserts in cis and in trans when

expressed from virus gene-vectors (Everett et al., 2010). For all its features as a pathogenicity

factor, evidence is lacking to suggest SPMV CP is a suppressor of gene silencing (Qiu and

Scholthof, 2004).

Satellite viruses are molecular parasites that depend on their helper viruses for replication

and movement in host plants (Scholthof et al., 1999; Hu et al., 2009). Our previous genetic and

molecular characterization of PMV and SPMV in millets revealed multiple determinants of the

synergism (Desvoyes and Scholthof, 2000; Turina et al., 2000; Qiu and Scholthof, 2001; Qiu and

Scholthof, 2004; Omarov et al., 2005; Batten et al., 2006a; Batten et al., 2006b; Everett et al.,

2010). In the case of PMV+SPMV mixed infections, SPMV has the unusual feature of enhancing

PMV accumulation in host plants, with a simultaneous increase in accumulation of PMV

movement protein (P8) and exacerbation of symptoms on systemically infected (non-inoculated)

leaves compared to infections of PMV alone (Scholthof, 1999) (Figure S1). These effects reflect

the historical definition of viral synergism: the mixed infection causes more severe symptoms

than a single infection and tends to facilitate an increase in the titer of one of the virus pairs

during the acute stage of the disease (Rochow and Ross, 1955; Latham and Wilson, 2008; Syller,

2012). However, the PMV+SPMV synergism stands unique because it involves a satellite virus.

Despite our past progress, the lack of a genetic model plant species made it challenging to study

the mechanism(s) underlying this synergism and effects on host plant processes.

Recently, Brachypodium was developed as a genomic model for studies of grass biology

and host-pathogen studies (Brkljacic et al., 2011). Brachypodium genome is syntenic to rice and

sorghum genomes and is related to several forage, small grains, and bioenergy crop plants



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including wheat, barley, and millets, all of which are in the grass subfamily Pooideae (Powell,

1994; The International Brachypodium Initiative, 2010). These temperate and cool season

grasses belonging to the Poaceae are the primary source of calories throughout the world

(Draper et al., 2001; Huo et al., 2006; Vogel et al., 2006; Vogel and Hill, 2008; Bevan et al.,

2010; Garvin et al., 2010; The International Brachypodium Initiative, 2010; Brkljacic et al.,

2011; Cao et al., 2011). In this study, we established Brachypodium as a systemic host for PMV

and SPMV infections, and show that the disease etiology mimics biological features previously

described in millet plants. Further, using microarray techniques, we have identified host

pathways affected by PMV and PMV+SPMV co-infections. From this, we have sketched a

model that shows the contributions of SPMV as a molecular effector of the helper virus, PMV,

and provide a more utilitarian definition of viral synergism that is based not only on the visual

symptoms but also on the altered host molecular processes that occur in viral synergism.

RESULTS

PMV and SPMV infection of Brachypodium results in a viral synergism

To study the synergism associated with PMV and SPMV infections, 1-week-old

Brachypodium plants with 2-3 leaves were inoculated with full-length infectious cDNA

transcripts of PMV alone or PMV+SPMV (Batten et al., 2006b). The PMV infection induced

chlorosis and necrosis of leaves with moderate stunting (Figures 1A, B), in comparison to mock

inoculated plants. In contrast, PMV+SPMV co-infection exacerbated the symptoms with

excessive stunting, severe chlorosis and necrosis, and reduced or lack of seed-set (Figures 1A,

B). Moreover, in the PMV+SPMV-infected plants, PMV CP was consistently detected several

days earlier than in plants inoculated with PMV alone (Figures 1D, E). Each of these features

exemplifies those we previously described in PMV+SPMV-infected millet plants (Scholthof,

1999).

To analyze the temporal progression of the disease in detail, and gain consistency in

comparisons among independent infections performed at different times, we classified PMV and

SPMV infections into three different stages based on the degree of visible symptoms and the

abundance of the viral proteins in the infected plants. Infected plants at stage I (typically ≤ 7 dpi)

showed no visible chlorosis symptoms on the inoculated leaves and had no detectable levels of

viral capsid proteins compared to mock-inoculated controls (Figures 1C, D, and E ). Infected



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plants at stage II (typically 10-14 dpi) displayed milder chlorosis and necrosis symptoms on both

the inoculated and newly emerging non-inoculated leaves and possessed detectable levels of viral

capsid proteins. Finally, stage III infected plants (typically ≥21 dpi) were severely chlorotic and

necrotic, were stunted, and had higher accumulation of viral capsid proteins (Figures 1C, D, and

E). With regards to developmental timeline of the infected plants, stage I and II plants are in their

early and late vegetative phase, respectively, while, stage III plants have transitioned to

reproductive phase.

Stage II chlorotic symptoms on upper non-inoculated leaves of PMV+SPMV-infected

plants typically appear by 10 dpi and become prominent by 21 dpi (stage III, Figure 2A, B). We

determined that the chlorotic leaves had reduced levels of chlorophyll A and chlorophyll B in the

PMV+SPMV-infected shoots (Figure 2C), compared to the mock-inoculated plants. Plants

infected with PMV+SPMV at maturity (stage III) also had significantly reduced biomass, based

on fresh and dry weights (Figure 2D). Additionally, PMV+SPMV-infected plants at stage III

were severely stunted (Figure 2B) with shorter internodes and smaller leaves (Figure 2E, F),

although tiller number was not affected (Figure 2G), when compared to mock-inoculated plants.

Seed set was also greatly or completely abolished in PMV+SPMV-infected Brachypodium at

stage III (Figure 1A, 1C), which we also described previously for PMV+SPMV infections in

millet plants (Scholthof, 1999). Finally, immunoblot analysis of the PMV+SPMV-infected plants

at stage II, detected both PMV CP and SPMV CP in upper non-inoculated leaves and root tissues

demonstrating systemic movement of both viruses (Figure 2H). Taken together, these results

show Brachypodium is a good model host plant for studying PMV and its SPMV-associated

synergistic interactions.

Transcriptome analysis of PMV and SPMV infected Brachypodium

To identify host molecular pathways altered by PMV and SPMV leading to the disease

symptoms, we performed genome-wide gene expression analysis using DNA oligonucleotide

microarrays. Because our intent was to identify the altered host molecular processes that

preceded the overall disease symptoms of the infected plants, we chose to analyze infected plants

before prominent visual chlorosis, necrosis, and stunting symptoms appeared. Above-ground

shoots (inoculated and non-inoculated leaves) of plants infected with PMV alone or co-infected

with PMV+SPMV, with mild chlorosis symptoms (stage II), along with mock-inoculated plants,



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were harvested for RNA isolation and microarray analysis. A custom-designed Affymetrix tiling

array, representing approximately 27,000 genes, by multiple probes, was used to assay three

independent biological replicates from each of the inoculation treatments. Normalized

microarray expression data was further corrected for false discovery rate (FDR) using the

Benjamini and Hochberg method (q-value <0.05) (Benjamini and Hochberg, 1995). In PMV-

infected plants, approximately 464 and 326 genes were up- and down-regulated by two-fold or

greater, respectively, in comparison to mock inoculated plants (Figure 3A). In PMV+SPMV co-

infection, 621 and 433 genes were up- and down-regulated by two-fold or greater, respectively

(Figure 3A). To validate the microarray results, we selected twenty candidate genes including

multiple protein kinases, transcription factors and metabolic enzymes (Table S1) and performed

quantitative (q) RT-PCR analysis using the same RNA used for the microarray analysis. The

criteria for choosing the twenty genes for (q) RT-PCR analysis was to represent genes in diverse

functional categories and with varying levels of mis-expression in the PMV and PMV+SPMV

infections (2 to 50-fold). The results of the (q) RT-PCR analysis strongly correlated to the

microarray data (Pearson correlation coefficient, r >0.9 and coefficient of determination, R2 >0.8,

Tables S1).

Next, we performed functional annotation of genes that were commonly induced or

repressed in PMV and PMV+SPMV infections using gene ontology (GO) terms for biological

processes (GO-P), molecular function (GO-F), and cellular component (GO-C) using the

AgriGO analysis tool (Du et al., 2010). To maximize functional annotations for more genes,

additional protein families (Pfam) (Punta et al., 2012) and protein analysis through evolutionary

relationship (PANTHER) (Thomas et al., 2003) functional annotations were assigned using the

Phytozome resource (Goodstein et al., 2012) (http://www.phytozome.net/). Further, gene set

enrichment was performed using the AgriGO singular enrichment analysis (SEA), with

appropriate statistical corrections (FDR<0.05). SEA identifies significant GO terms that are

overrepresented in a query gene list relative to a pre-calculated frequency in the reference (or

background) genome of a species and allows a general inference of the overrepresented

biological processes or GO terms. The SEA revealed that both PMV alone and PMV+SPMV

infections altered expression of multiple genes in biological processes such as primary

metabolism, metabolite transport, defense, and cell wall remodeling (summarized in Figure 3B

and expanded details in Table S2 ), suggesting that PMV alone and SPMV-synergistic



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interactions altered the metabolic fate of the infected cells. Further, among the induced genes,

several detoxification and stress-responsive transcripts corresponding to glutathione S-

transferases, cytochrome P450, chaperone and heat shock proteins, ribosomal proteins, and

proteasome components were overrepresented (Figure 4A and Table S2). Interestingly, multiple

nuclear-encoded, photosynthesis-related chlorophyll A/B binding and photosystem proteins were

predominant among the down-regulated genes (Figure 4B and Table S2). The latter result lends

genetic support for our observations of the leaf chlorosis (Figure 1A, 2A) and decreased

chlorophyll levels (Figure 2C) in the PMV+SPMV infected shoots.

Host defense responses to PMV and SPMV

Because high-quality genome sequence information is available for Arabidopsis, and

because antiviral defenses are well-studied in that model (Whitham et al., 2003; Ascencio-Ibáñez

et al., 2008; García-Marcos et al., 2009; Elena et al., 2011; Hanssen et al., 2011), for our

subsequent analysis, we chose to compare our transcriptome results with Arabidopsis-virus

responses. Moreover, such a comparison allows us to compare and contrast antiviral responses in

monocot and dicot plants. Among the altered antiviral silencing components, transcript levels of

a SDE3-like helicase (Bradi3g52690), which in Arabidopsis is involved in secondary

amplification of silencing and siRNA production (Dalmay et al., 2001), was suppressed in PMV

and PMV+SPMV infections by ≥2-fold compared to mock-inoculated plants (Table S1). In

Arabidopsis, activation of pathogenesis-related (PR) proteins is a classical marker for basal and

induced defense responses involving salicylic acid (SA), pathogen-triggered immunity (PTI),

effector-triggered immunity (ETI), and systemic acquired resistance (SAR) (Pieterse et al., 2009;

Vlot et al., 2009; Padmanabhan and Dinesh-Kumar, 2010; An and Mou, 2011). Among the PMV

and PMV+SPMV modulated genes in Brachypodium that are functionally annotated as stress or

stimulus responsive (Figure 3B), two cysteine-rich secretory proteins (Bradi1g57580 and

Bradi1g57590) were induced greater than twenty-fold compared to mock controls (Table I). We

identified these as PR-related proteins and, in Arabidopsis, were closest to PR-1. Further

candidate gene searches revealed increased expression of several PR genes orthologous to

Arabidopsis PR-2, PR-3, PR-4, and PR-5 (Table I) in PMV alone and PMV+SPMV co-infection.

Interestingly, multiple Brachypodium PR genes that were strongly induced in PMV infection

were relatively less induced in the PMV+SPMV co-infection (Table I). For example, expression



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of a PR-1-like gene was up-regulated by 40-fold in PMV alone infection compared to mock,

however, its expression was induced only 20-fold in PMV+SPMV infections. Similar expression

patterns were observed for other PR genes (Table I). Because PMV and PMV+SPMV infection

induced multiple PR genes, we next determined whether other components of SA signaling were

altered. We found upregulation of ISOCHORISMATE SYNTHASE 1 (ICS1)-like, ABERRANT

GROWTH AND DEATH 2 (AGD2), ALTERNATIVE OXIDASE (AOX), and certain WRKY

factors, while expression of a PHYTOALEXIN DEFICIENT4 (PAD4)-like gene was down-

regulated (Table I). The closest orthologs of each of these gene products in Arabidopsis are

implicated in SA biosynthesis and signaling (Vlot et al., 2009; An and Mou, 2011).

Activation of PR genes by SA or pathogen infection is likely a downstream secondary

response, mediated in Arabidopsis by NPR1, TGA, and WRKY-family transcriptional-regulators

(Moore et al., 2011). From our microarray results, transcript levels of NPR1 and TGA family

members were not affected by PMV or PMV+SPMV infection (data not shown). However,

multiple WRKY factors were up-regulated in PMV and PMV+SPMV infections (Table I and

Figure 5). WRKY factors belong to a super-family of DNA-binding transcriptional regulators

which are crucial for plant growth and development, and mediate responses to biotic and abiotic

stress and innate immunity (Rushton et al., 2010). To identify PMV- and PMV+SPMV-affected

WRKY factors, we performed multiple-sequence alignment (MSA) and phylogenetic

comparisons with Arabidopsis WRKY factors. In Arabidopsis, WRKY factors are classified into

different groups based on their WRKY and zinc-finger domains (Rushton et al., 2010). As

expected, Brachypodium WRKY factors modulated in PMV and PMV+SPMV infection have

signature WRKY and zinc-finger motifs (Figure 5A), and belonged to group IC (Bradi2g05510),

group IIa (Bradi4g30360), group IIc (Bradi4g19060), and group III (Bradi2g15877 and

Bradi2g30695) (Figure 5B). In Arabidopsis, WRKY18, -53, -54, and -70 have roles in SA-

mediated responses to bacterial and fungal diseases (Wang et al., 2006). PMV- and SPMV-

modulated WRKY factors were phylogenetically closer to Arabidopsis WRKY10 (group IC);

WRKY18, -40, and -60 (group IIa); WRKY75 (group IIc); and WRKY53, -41, -54, and -70

(group III), respectively (Figure 5B), suggesting their potential roles in mediating SA responses

in PMV or PMV+SPMV infection.



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PMV and SPMV modulate expression of jasmonic-acid and ethylene signaling related

genes

In addition to SA, two other hormones, jasmonic acid (JA) and ethylene (ET), play

important roles in plant defenses against pathogens (Pieterse et al., 2009; Verhage et al., 2010;

An and Mou, 2011). Although there are reports of SA and JA synergy (Mur et al., 2006), in

general SA antagonizes JA and ET responses, especially in mediating defense against biotrophic

pathogens (Vlot et al., 2009). PMV and PMV+SPMV infections suppressed the expression of

putative Brachypodium LIPOXYGENASE2 (LOX2) and ALLENE OXIDE SYNTHASE (AOS)

enzymes (Table II), which in Arabidopsis are associated with JA biosynthesis. Although

expression of 12-OPDA REDUCTASE3 (OPR3), an enzyme functioning downstream of LOX2

and AOS in JA biosynthesis was up-regulated (Table II), two putative JA-responsive genes

including VEGETATIVE STORAGE PROTEIN1 (VSP1) and FATTY ACID DESATURASE7

(FAD7) were down-regulated (Table II). Few of the PMV and SPMV infected plants transitioned

to flowering (Figure 1)—these plants had fewer flowers, reduced or no seed-set, and poor

viability (Figure 1, and data not shown). Such reproductive defects are also common in several

JA-deficient mutants, such as fad3/fad7/fad8, aos, and opr3 in Arabidopsis (Wasternack, 2007),

and correlate to the suppressed JA responses in the PMV and SPMV infected plants.

The precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC), is synthesized

by ACC synthase (ACS). Production of ACC is a rate-limiting step in ethylene biosynthesis.

ACC is further converted to ethylene by ACC oxidase (ACO) (Dong et al., 1992). PMV- and

PMV+SPMV-infected plants had higher levels of ACO1 transcript compared to mock (Table II).

However, expression of several putative ethylene-response factors such as ETHYLENE

RESPONSE FACTOR-1 (ERF-1), ERF3-like, ERF4, and ERF/Related to AP2.2 (RAP2.2) were

down-regulated (Table II), suggesting suppressed ET signaling in the infected plants.

Together, these results support alteration of JA and ET signaling, as a component of host

immune responses to PMV and PMV+SPMV infection, possibly via interactions with SA

signaling (Vlot et al., 2009).

Temporal expression analysis of putative SA, JA and ET components during the

progression of PMV and SPMV infection

Because the altered SA, JA and ET responses in the PMV and PMV+SPMV-infected



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plants identified using microarray analysis was only a snapshot of the affected defense responses

before the onset of severe disease symptoms (stage II), we analyzed their dynamics during the

other stages of infection. For this, we sampled PMV and PMV+SPMV-infected plants when they

displayed no symptoms (stage I), mild chlorosis symptoms (stage II), and severe chlorosis,

necrosis, and stunting symptoms (stage III), as described earlier (Figure 1). Next, we performed

(q) RT-PCR analysis to quantify expression of putative SA (PR-1-, PR-3-, PR-5-, PAD4-like and

AOX1A), JA (LOX2, AOS and OPR3) and ET (ERF-1 and ERF3-like) components in the infected

plants. Based on the microarray data (Table I and II), the aforementioned genes demonstrated

varying levels of misexpression in PMV and PMV+SPMV-infected plants. The temporal

expression analysis revealed activated expression of PR-1, PR-3, and PR-5-like genes in PMV

(≥2.8-fold) and in PMV+SPMV-infected plants (≥1.7-fold), when compared to mock-inoculated

plants as early as stage I, when visible symptoms were not evident (Figures 6A-C). PR gene

expression continued to increase in subsequent stages of infection (stage II and stage III)

(Figures 6A-C). Also consistent with the microarray data, expression of multiple PR genes was

significantly less induced in PMV+SPMV-infected plants compared to PMV alone infection

(Figures 6A-C and Table I). Similarly, expression of AOX1A was induced ≥250-fold in PMV

and ≥50-fold in PMV+SPMV-infected plants at stage I and was ≥500-fold at stages II and III

(Figure 6D) in both infections. In contrast to the activated expression of PR and AOX genes that

are components downstream of SA signaling in Arabidopsis, expression of a putative PAD4-like

gene, an upstream positive regulator of SA responses (Zhou et al., 1998), was significantly

downregulated in the infected plants in stages II and III; however, its expression was slightly

induced (~1.3 to 1.4-fold; p-value ≤0.05) early in the infection (stage I). Further, expression of

multiple JA and ET components LOX2, AOS, ERF-1, and ERF3-like genes were all uniformly

suppressed in PMV and PMV+SPMV-infected plants, while expression of OPR3 was

upregulated (Figure 6F-J)—results that are again consistent with the microarray data. Similar to

the expression pattern of the PAD4-like gene, expression of LOX2 and ERF3-like genes were

significantly higher early in the infection (stage I), but were suppressed in stages II and III when

compared to the mock-inoculated plants (Figures 6D, 6G, 6J). Moreover, multiple SA, JA, and

ET components in Brachypodium were developmentally regulated. PR-1, PR-3 and PR-5-like

gene expression was highest in plants in their reproductive phase with 2.3, 2.0, and 2.3-fold

change induction, respectively, when compared to younger plants at stage I (Figures 6A, 6B and



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6C). Expression of AOS and ERF-1 was also higher early in the vegetative phase (stage I), while

their expression was suppressed in late vegetative phase (stage II), as well as in the reproductive

phase (stage III) (Figures 6F and 6I). LOX2 and ERF3-like genes displayed the opposite pattern,

with lowest expression early in the vegetative phase (stage I) (Figures 6G and 6J). Interestingly,

PMV and SPMV infection perturbed the normal developmental expression pattern of LOX2 and

ERF3-like genes, but, not AOS and ERF-1 (Figures 6F, 6G, 6I and 6J).

Together, these analyses not only corroborate the microarray findings, but also reveal the

dynamic expression patterns of multiple SA, JA, and ET components during Brachypodium

development, and their differential perturbation in PMV and SPMV infections.

PMV and SPMV infection modulate expression of multiple receptor-like kinase/Pelle

members

PMV- and PMV+SPMV-infection altered expression of several serine-threonine kinases

(STKs). Expression of twenty-two STKs was up-regulated by two-fold or greater (FDR <0.05) in

PMV and PMV+SPMV infection (Table III), with six additional STKs whose expression was

affected by 1.5 to 2-fold (data not shown). Based on the predicted protein sequences and the

functional domains, the majority of these kinases were identified as being receptor-like

kinase/Pelle (RLK) proteins (Shiu et al., 2004). Most plant RLKs are transmembrane proteins or

membrane-associated proteins with intracellular kinase domains, although some are localized to

the cytosol (Shiu et al., 2004; Padmanabhan and Dinesh-Kumar, 2010). Using MSA analysis and

phylogenetic comparisons, we identified PMV- and PMV+SPMV-affected kinases as belonging

to RLK subfamilies LRR-VIII-2, LRR-XII , WAK/LRK10L-1, RLCK-OS2, RLCK-IV, RLCK-

IXb, SD-1c , SD-2b, L-LEC, and DUF26, in addition to non-RLK subfamilies such as

MEKK_ste11_MAP3K , KIN1/SNF1/Nim1_like (CAMK_2), PVPK_like (AGC_8)_kin82y

(Figure 7 and Table III). Several of the RLKs possessed additional domains such as leucine rich

repeat (LRR), calcium-binding epidermal growth factor (EGF), legume-lectin (L-LEC), D-

mannose binding lectin, and U-box domains (Figure 7), and three RLKs possessed domains of

unknown function (DUF26). Kinases such as those possessing L-LEC, D-mannose binding

lectin, and DUF26 also contained putative transmembrane domains (Table S3), suggesting they

may function as transmembrane RLKs. The general up-regulation of several host kinases and

RLKs suggests that phosphorylation modifications of either host or virus-encoded proteins are



Mandadi, p. 16

crucial signals in PMV and PMV+SPMV infection.

The SPMV synergism: modulation of PMV-triggered defense gene expression and

alteration of unique nuclear factors

Because SPMV enhanced PMV accumulation, and exacerbated disease symptoms (Figure

1 and (Scholthof, 1999), we analyzed the effect of SPMV on host molecular processes.

Unexpectedly, multiple genes in SA, JA, and ET defenses, particularly PR and AOX genes that

were altered in PMV single infections, consistently demonstrated lower fold-changes of

misexpression in co-infection with SPMV, as determined by microarray and (q) RT-PCR

analysis (Table I, II, and Figure 6). In contrast to the antagonistic effect on defense gene

expression, a large number of genes in diverse functional categories displayed additive or

synergistic alteration in their expression patterns during a co-infection of SPMV (Table IV and

Table V). Intriguingly, few distinctive genes were uniquely altered by SPMV (Table VI),

compared to PMV alone or mock-inoculated plants. After verification by (q) RT-PCR, we

identified these unique up-regulated genes as spliceosome-associated factors (Bradi2g09850 and

Bradi5g07850), an AP2-family transcription factor (Bradi3g06562), and a protein of unknown-

function (Bradi4g34700) with a predicted nuclear-localization signal (based on PSORT analysis,

data not shown).

Taken together, these results show that SPMV modulates PMV-triggered defense gene

expression and uniquely activates expression of putative nuclear-localized proteins involved in

transcription and splicing. We hypothesize that the cumulative effect of these processes relates to

the exacerbated disease symptoms induced by SPMV in PMV+SPMV co-infections.

DISCUSSION

In contrast to dicot plant virus research, studies with monocot-infecting viruses have

lagged due to the lack of a reliable genetic model. Grasses, including rice, maize, switchgrass,

wheat, and sorghum have long lifecycles, require an abundance of greenhouse space, and

oftentimes lack critical genomic tools (microarrays, annotated genomes, etc.). Recently,

Brachypodium has emerged as a promising model for grass biology research (Draper et al., 2001;

Huo et al., 2006; Vogel et al., 2006; Vogel and Hill, 2008; Bevan et al., 2010; Garvin et al.,

2010; The International Brachypodium Initiative, 2010; Brkljacic et al., 2011; Cao et al., 2011).



Mandadi, p. 17

Here, we demonstrated the utility of Brachypodium as a model system for plant pathobiology

studies with a focus on the synergism of the mixed infection of PMV and SPMV. PMV- and

PMV+SPMV-infected Brachypodium produced the same disease outcome we had previously

observed with various millet species (Scholthof, 1999), including severe symptoms of chlorosis

and necrosis, stunting, and reduced seed-set (Figures 1 and 2). The earlier detection of PMV CP

together with the exacerbated symptoms of PMV+SPMV-infected plants compared to PMV

alone infection (Figure 1) supports a role for SPMV in enhancing PMV accumulation in

Brachypodium, a finding consistent with our previous reports of PMV+SPMV synergism in

millets (Scholthof, 1999). Because viral synergistic interactions can be host-specific (González-

Jara et al., 2004), recapitulating the results of these earlier experiments in millet species was a

critical first step towards establishing Brachypodium as a robust genetic model species for

studying PMV and SPMV interactions.

Our comparative transcriptome analyses of PMV alone and the mixed PMV+SPMV

infections in Brachypodium (summarized in Figure 8) with previously known dicot-infecting

plant viruses (Whitham et al., 2003; Ascencio-Ibáñez et al., 2008; García-Marcos et al., 2009;

Elena et al., 2011; Hanssen et al., 2011) revealed several distinctive features of virus-host

interactions of monocots. PMV- and PMV+SPMV-infection prominently triggered salicylic acid

(SA)-mediated defense responses, a response that appears conserved with several dicot host:virus

interactions (Murphy et al., 1999; Kachroo et al., 2000; Whitham et al., 2003; Huang et al., 2005;

Whitham et al., 2006; Ascencio-Ibáñez et al., 2008; Hanssen et al., 2011; Pacheco et al., 2012).

Multiple genes in the pathogenesis-related (PR) protein family were strongly up-regulated in

PMV alone and PMV+SPMV infections in Brachypodium (Table I and summarized in Figure 8).

Brachypodium has two PR-1-like (Bradi1g57580 and Bradi1g57590) and two PR-5-like

(Bradi1g13060 and Bradi1g13070) genes that are tandemly arranged on chromosome 1.

Expression of both copies of PR-1-like and PR-5-like genes was induced in PMV and

PMV+SPMV infections (Table I), suggesting that these genes are transcriptionally co-regulated.

Similarly, expression of three tandemly arranged subunits of AOX, an enzyme activated by SA

and pathogen infection (Lennon et al., 1997), was uniformly up-regulated (Table I and

summarized in Figure 8). Although Arabidopsis also has tandemly arranged PR and AOX-genes,

it is not known if they are transcriptionally co-regulated in response to pathogen infections. PR

gene expression was also activated in healthy Brachypodium plants (mock-inoculated) as they



Mandadi, p. 18

matured (Figure 6). This correlates with their known roles in Arabidopsis developmental leaf

senescence (Lim et al., 2007)—a process triggered as plants mature. PMV and SPMV infection,

however, constitutively activated PR-1, PR-3, PR-5-like genes and AOX 1A, although expression

of PR3-like gene and AOX1A displayed a trend of downregulation as the infected plants matured

(Figure 6). Despite the strong activation of PR genes and other SA components, PMV and

PMV+SPMV infected plants did not display precocious senescence (data not shown), suggesting

that the role of PR proteins in defense responses are distinct from their function in developmental

senescence.

In support of the antagonistic relationship between SA and JA/ET responses, PMV and

PMV+SPMV infection suppressed expression of multiple JA and ET components (Table II,

Figure 6 and summarized in Figure 8). JA and ET hormones are also implicated in plant

development, including senescence (van der Graaff et al., 2006) and flower development

(Wasternack, 2007). In Arabidopsis, expression of LOX2 is induced in response to wounding

(Bell et al., 1995), methyl-jasmonate treatment (Jensen et al., 2002) and senescence (van der

Graaff et al., 2006). Here, we found that the expression of LOX2 was developmentally regulated,

with highest expression in mature plants (Figure 6). Expression of AOS, an enzyme functioning

downstream of LOX2 in JA biosynthesis (Wasternack, 2007), was also developmentally

regulated; however, its expression appeared antagonistic to LOX2, with lowest expression in the

mature plants (Figure 6).

Although the cross-talk between JA and ET signaling in plant-pathogen interactions is

widely known, relatively less information is available regarding the transcriptional regulation

and feedback between the two pathways. In Arabidopsis, members of AP2/ERF family of

transcription factors, such as ERF1, mediate JA-induced transcriptional changes in response to

pathogen infections, as well as, to exogenous methyl-jasmonate or ethylene (Lorenzo et al.,

2003). In Catharanthus roseus (periwinkle), an AP2/ERF-domain transcription factor, ORCA3,

binds directly to the jasmonate- and elicitor-responsive element (JERE) in the promoter of a

terpenoid indole alkaloid biosynthetic gene, STRICTOSIDINE SYNTHASE (STR), and activates

its expression in response to methyl-jasmonate (van der Fits and Memelink, 2001). PMV and

PMV+SPMV infection modulated expression of multiple AP2/ERF-like transcription factors,

including ERF-1 and ERF3-like genes (Table II and S4). Moreover, expressions of ERF-1 and

ERF3-like gene appeared to be developmentally regulated (Figure 6), and interestingly,



Mandadi, p. 19

paralleled the expression patterns of AOS and LOX2, respectively (Figure 6). Given the role of

ERF proteins in transcriptional regulation of JA components (van der Fits and Memelink, 2001;

Lorenzo et al., 2003), it is tempting to speculate the Brachypodium ERF-1 and ERF3-like factors

we identified here, could mediate AOS and LOX2 expression. Nevertheless, the simultaneous

suppression of JA and ET components in PMV and PMV+SPMV infection, and the overlapping

developmental expression patterns of JA-biosynthesis genes and ERF transcription factors,

supports transcriptional co-regulation of JA and ET components.

In contrast to the prominent changes in expression of SA, JA and ET defense components,

components of post-transcriptional gene silencing (PTGS) defenses were largely unaffected in

PMV and PMV+SPMV infections. PTGS via short-interfering RNA (siRNA) is a conserved

mechanism of defense against viruses (Dunoyer and Voinnet, 2005; Alvarado and Scholthof,

2009; Alvarado and Scholthof, 2012). For example, tomato plants infected with Pepino mosaic

virus (PepMV), a Potexvirus, activates multiple Dicer-like (DCL) and AGO genes during the

infection (Hanssen et al., 2011). Similarly, Arabidopsis DRB4 expression is induced upon

infection of Turnip yellow mosaic virus (TYMV) (Jakubiec et al., 2012) and expression of RDR1

is up-regulated in tobacco plants infected with Tobacco mosaic virus (TMV) (Xie et al., 2001)

and PVY (Rakhshandehroo et al., 2009). Brachypodium has 7 DCL, 4 DRB, 2 HEN, 14 AGO, 6

RDR, and 3 SILENCING DEFECTIVE 3 (SDE3)-like genes (data not shown). In contrast to the

PepMV, TYMV, and PVY responses (Rakhshandehroo et al., 2009; Hanssen et al., 2011;

Jakubiec et al., 2012), expression of DCL, DRB, AGO, HEN, and RDR genes in Brachypodium

was not affected by PMV or PMV+SPMV infections (data not shown), although we cannot rule

out changes in protein level or activity or changes that occur transiently. However, expression of

an SDE3-like helicase was suppressed in both PMV and PMV+SPMV infections (Table S1). It is

possible that a PMV-encoded protein functions to suppress SDE3 expression. Nevertheless, not

activating (or otherwise evading) antiviral RNA silencing surveillance, and suppressing SDE3

may be a strategy used by PMV and SPMV to enable productive infection in Brachypodium and,

perhaps, in the other temperate grasses that support this mixed infection (Buzen et al., 1984;

Scholthof, 1999).

Several transcriptional regulators belonging to TGA, WRKY, and MYB gene families

orchestrate SA-mediated transcriptional changes, including activation of PR and other defense-

related genes, as well as modulate JA and ET signaling and influence plant defenses (Moore et



Mandadi, p. 20

al., 2011). In Arabidopsis, SA-mediated defense responses are either NPR1-dependent or

independent (An and Mou, 2011). Both SA and pathogen infection induce expression of NPR1,

in part via the WRKY transcription factors that bind to NPR1 promoter-proximal “W-box”

sequences (TTGACC/T) (Yu et al., 2001). Moreover, NPR1 physically interacts with TGA

factors within the nucleus and its localization/activity is post-translationally regulated by SA

(Tada et al., 2008). Although Brachypodium NPR1 (Bradi2g05870) also contains two putative

W-box elements within a 50 bp region upstream of the transcriptional start site (-242 to -292 bp),

its expression was not altered in PMV or PMV+SPMV infections (data not shown), suggesting it

to be less likely a transcriptional target of the WRKY factors mis-expressed in PMV- and

PMV+SPMV-infections (Figure 5 and Table S4). Moreover, we did not find alterations in the

expression of TGA factors in PMV or PMV+SPMV infections (data not shown). Together, these

results suggest that PMV- and SPMV-induced defense responses may be NPR1-independent.

Although we do not know if BdNPR1 activity or sub-cellular localization was affected, our

results agree with reports in Arabidopsis compatible host-virus interactions, whereby expression

of the majority of defense-related genes are NPR1-independent (Kachroo et al., 2000; Huang et

al., 2005).

Whitham et al. (Whitham et al., 2003) conducted the most comprehensive analyses of

profiling genome-wide expression changes in response to diverse RNA virus infections in

Arabidopsis. Among the genes altered in several functional categories, five protein kinases,

putatively involved in signal transduction, were differentially expressed in Arabidopsis infected

with Cucumber mosaic virus (genus Cucumovirus), Oil seed rape mosaic virus (genus

Tobamovirus), Turnip vein clearing virus (genus Tobamovirus), PVX (genus Potexvirus) and

Turnip mosaic virus (genus Potyvirus) (Whitham et al., 2003). In comparison to these compatible

Arabidopsis viral responses, and other compatible dicot host:virus responses (Murphy et al.,

1999; Kachroo et al., 2000; Whitham et al., 2003; Huang et al., 2005; Whitham et al., 2006;

García-Marcos et al., 2009; Hanssen et al., 2011), PMV and PMV+SPMV infections in

Brachypodium altered expression of a higher proportion of protein kinase genes (27 kinases)

(Table III; SEA FDR<0.05, Table S2), predominantly in the RLK/Pelle family (Shiu et al.,

2004). These RLKs possessed additional LRR, WAK/calcium-binding EGF, L-LEC, D-mannose

binding lectin, and DUF26 domains (Table III and Figure 7), and several of them contained

putative transmembrane domains and W-box elements in their promoter-proximal sequences



Mandadi, p. 21

(Table S3). Together, we suggest that these kinases are likely candidates to function as

transmembrane RLKs and are putative targets of WRKY transcription factors induced in PMV

and PMV+SPMV infections.

To our knowledge, few compatible monocot host:virus responses have been studied using

transcriptome tools compared to dicot host:virus responses. Our finding of several up-regulated

kinases in PMV- and PMV+SPMV-infected Brachypodium are comparable to responses found

in maize infected with Rice black-streaked dwarf virus (RBSDV; genus Phytoreovirus) (Jia et

al., 2012) and rice infected with Rice stripe virus (RSV; genus Tenuivirus) (Satoh et al., 2010),

but not to Rice dwarf virus (RDV; genus Phytoreovirus) infections in rice (Shimizu et al., 2007).

RBSDV and RSV infections in maize and rice causes mis-expression of fourteen and forty-two

protein kinase genes, respectively (Satoh et al., 2010; Jia et al., 2012), although the proportion of

RLKs and their characteristics are unclear. The rice genome encodes a substantially greater

number of kinases (kinome size: ~1454), compared to Brachypodium (kinome size: ~1177) and

Arabidopsis (kinome size: ~1027 kinases) (Shiu et al., 2004; Dardick and Ronald, 2006; The

International Brachypodium Initiative, 2010). Interestingly, the RLK/Pelle family is predicted to

be larger even in the common ancestor of Arabidopsis and rice which split ca. 150-200 million

years ago and continued to undergo lineage-specific expansions in monocots and dicots alike

(Shiu et al., 2004; The International Brachypodium Initiative, 2010). The large number of RLKs

among the kinases in general may explain the relatively higher abundance of RLKs (24 out of

27) in the “PMV/SPMV kinome” when compared to the other kinase sub-families (Figure 7).

However, because the kinome sizes of Brachypodium and Arabidopsis are comparable, and

because RLKs underwent parallel expansions in both dicots and monocots, mere differences in

kinome size or composition does not explain why a higher proportion of kinases are mis-

expressed in PMV and PMV+SPMV infected Brachypodium when compared to other dicot:virus

infections. Perhaps mis-expression of these protein kinases is a unique feature of monocot

host:virus interactions, with biological significance in antiviral defenses.

Plant RLKs are either cytosolic (e.g., RLCKs) or membrane-spanning (e.g., receptor

histidine kinases) and mediate growth and development by serving as receptors to hormones

such as ethylene (Bleecker and Kende, 2000), cytokinin (Higuchi et al., 2004), and

brassinosteroids (Li and Chory, 1997). Some RLKs also recognize pathogen associated

molecular patterns (PAMPs), such as flagellin, chitin, or effector proteins involved in plant



Mandadi, p. 22

innate immune responses—commonly referred to as pathogen recognition receptors (PRR)

(Afzal et al., 2008; Dodds and Rathjen, 2010). In Arabidopsis and rice, fewer than 20% of all

RLKs constitute PRRs (e.g., Arabidopsis flagellin-sensitive FLS2 (Gómez-Gómez and Boller,

2002) and rice XA21 (Chen et al., 2010)). PRRs typically lack an arginine (R) residue preceding

the invariant aspartic acid (D) in the catalytic site and are referred to as non-RD kinases (Chen et

al., 2010). In the PMV/SPMV kinome, Bradi5g02980, Bradi4g38690, Bradi4g21990, and

Bradi2g47510 are non-RD kinases (Table S3 and data not shown), suggesting that they could

function in a manner similar to PRRs. Despite the importance of PRRs in bacterial and fungal

diseases (Afzal et al., 2008; Dodds and Rathjen, 2010) their function and that of the other RLKs

in virus-host interactions are not known.

Unlike bacterial and fungal pathogens, plant viruses are mostly located within a cell and

use symplastic connections (plasmodesmata) for cell-to-cell movement. Moreover, plant viruses

do not produce PAMPs or encode effector-like proteins for cytoplasmic or cell-surface localized

RLK recognition (Carr et al., 2010), yet RLKs could function in signal transduction in viral

infection. Reflecting on this scenario, we suggest that RLKs (such as PRRs) could perceive

virus-associated signals. As summarized in Figure 8, RLKs could recognize PMV and SPMV

virions or their encoded proteins either at the plasmodesmata (via transmembrane RLKs) or in

the cytoplasm (via cytosolic RLKs). In support of this, recent proteomic studies have identified

several RLKs as an abundant class of membrane proteins enriched at the plasmodesmata

(Fernandez-Calvino et al., 2011; Jo et al., 2011). Alternatively, and in a manner similar to wall-

associated kinases (WAKs), RLKs may recognize damage associated molecular patterns

(DAMPs), such as oligogalacturonides (Brutus et al., 2010), released by the damaged cells as a

consequence of virus infection. Although these scenarios are not mutually exclusive, further

studies to determine the localization and interactions between PMV- and SPMV-encoded

proteins and the host protein kinases mis-regulated in the infection will test these hypotheses.

Synergism among viruses is generally described by enhanced host disease symptoms and

enhanced movement of the viruses as a mixed complex versus movement of individual viruses

(Rochow et al., 1955). The best known example of viral synergisms, the mixed infection of PVX

and PVY results in an increased accumulation of PVX by upwards of 40-fold in plants

permissive for systemic infection compared to single infections (Rochow and Ross, 1955),

primarily due to the effect of PVY-encoded silencing suppressor protein, HC-Pro (Pruss et al.,



Mandadi, p. 23

1997). Very little is known about the host processes affected by PVX-PVY synergism or viral

synergisms in general. Using an orthologous potato cDNA array, Garcia-Marcos (2009)

determined that PVX+PVY co-infection in N. benthamiana activated putative genes in JA

biosynthesis pathway (García-Marcos et al., 2009). In another study, expression of specific

microRNAs (miRNAs) such as miR156, 171, 398 and 168 were found to be differentially

expressed amongst single versus mixed infections of PVX , PVY, and Plum pox virus (PPV) in

N. benthamiana (Pacheco et al., 2012). However, the biological relevance of the miRNAs

(Pacheco et al., 2012) and the misregulated JA components (García-Marcos et al., 2009) in the

potyviral synergisms are not clear. Mechanistically, the SPMV synergism is unlike the potyviral

synergism that involves the HC-Pro silencing suppressor, as SPMV (i.e., SPMV CP) has no

known role in suppressing silencing (Qiu and Scholthof, 2001).

Although PMV accumulated to detectable levels earlier in the presence of SPMV in

Brachypodium (Figure 1) and in millets (Scholthof, 1999), the timing of the disease symptom

appearance of PMV and PMV+SPMV infected plants was similar, suggesting that SPMV has

only modest effect in altering the rate of infection or the disease progression. However, SPMV

infection did exacerbate the disease symptoms (Figure 1 and 2), and additively altered host gene

expression (Table IV and VI), in a manner similar to PVX-PVY synergistic interactions in N.

benthamiana (García-Marcos et al., 2009). We hypothesize that the early detection of PMV and

the exacerbation of symptoms could be a result of greater PMV accumulation per cell and/or

more cells infected with PMV in the presence of SPMV, possible due to SPMV enhancing PMV

replication (Batten et al., 2006b) and/or PMV movement. In contrast to the additive effects on

gene expression (Table IV and VI), expression of multiple SA and JA signaling genes were

relatively less mis-expressed in PMV+SPMV co-infection, when compared to an infection of

PMV alone (Table I, Figure 6). Moreover, SPMV uniquely modulated expression of few

transcription factors (Table VI). These observations suggest that at least some of the SPMV-

induced effects are not direct consequences of enhancing PMV accumulation. Based on the

altered gene expression data alone, it is difficult to ascertain if these additively or

antagonistically altered genes are a direct cause or consequence of the exacerbated disease

symptoms in PMV+SPMV infection, but they are indicative of the synergistic role imparted by

SPMV at the molecular level.

SPMV uniquely altered the expression of putative nuclear-localized proteins in splicing



Mandadi, p. 24

processes, a pre-mRNA splicing factor 3A subunit 1, and a splicing factor 1/branch-point

binding protein (Table VI), suggesting that SPMV may also function by affecting splicing-

related processes. A preliminary analysis of the microarray data detected significant changes

(FDR<0.05) in expression of alternatively spliced transcripts in select genes including an RNA

polymerase II subunit in PMV+SPMV infections compared to PMV alone (Table S5), although

the fold-change in expression of these transcripts was low. The alternatively spliced transcripts

originated from intron retentions or alternate splice-site preferences (Table S5) and altered the

primary protein sequence. Alternative splicing can influence plant defense responses, as

demonstrated for fungal, bacterial, and viral infections (Dinesh-Kumar and Baker, 2000; Jordan

et al., 2002; Zhang and Gassmann, 2003; Ayliffe et al., 2004; Reddy, 2007). For example,

alternative splicing of eukaryotic translation initiation factor 4E (eIF4E) was implicated in

tolerance of tomato against two potyviruses, PVY and Pepper mottle virus, but not to Tobacco

etch virus (also a Potyvirus) (Robaglia and Caranta, 2006; Piron et al., 2010). Although the

observed effects of SPMV on alternative splicing may be a consequence of exacerbated stress

inflicted by PMV/SPMV synergism, we suggest that SPMV CP may alter splicing-related

processes, based on our previous findings—SPMV CP is a bona fide RNA-binding protein,

stabilizes RNA virus gene inserts in cis and in trans, and translocates to the nucleolus (Qiu and

Scholthof, 2004; Qi et al., 2008; Everett et al., 2010). Further studies are needed to clarify the

exact mechanism(s) and biological significance of the alternatively spliced transcripts mis-

expressed in PMV+SPMV synergism.

Regardless of the exact mechanisms, the role of SPMV as a molecular effector to PMV

offers an explanation for the exacerbated symptoms in plants infected with PMV+SPMV

compared to PMV alone. By analogy to host RNA silencing processes that exert selective

pressure on viruses to acquire viral suppressors like HC-Pro, an evolutionary pressure on PMV

to acquire disease modulating effectors like SPMV may have primed the synergism. Taken

together our findings show that despite viral synergisms produce similar outcomes of

exacerbated disease symptoms at the phenotypic level on the host plants, they trigger unique and

defined changes at the molecular level. To our knowledge, this is the first report of a global gene

expression analysis of a plant virus synergism in Brachypodium and Poaceae in general.



Mandadi, p. 25

MATERIALS AND METHODS

Plant growth conditions and virus inoculations

Brachypodium distachyon inbred line Bd21 (Brachypodium) seeds were sown in Metro-

Mix 366 (SunGrow Horticulture), stratified at 4 °C for 3-4 days, and were subsequently

transferred to diurnal growth conditions of 14 h light (21 °C) /10 h dark (18 °C ) under a light

intensity of ~250-280 µmol/m2s with 60% relative humidity. To induce flowering, 6-week-old

plants were vernalized at 4 °C under continuous low light (20-30 µmol/m2s) for 3 weeks and

subsequently transferred to diurnal growth conditions. Alternatively, for viral infections, plants

were vernalized at germination by cold-treating the seeds at 4 °C for 7-10 days, followed by

growth in diurnal conditions. For viral inoculations, full-length infectious clones of PMV and

SPMV in pUC119 vectors were linearized using EcoICR1 and BglII restriction endonucleases,

respectively (Turina et al., 1998). Linearized plasmid DNA (500 ng) was used for in vitro

transcription reactions using T7 RNA polymerase (Fermentis) following manufacturer’s

instructions. After verification of the integrity of transcripts in agarose gels, approximately 100

ng of PMV and SPMV transcripts in RNA inoculation buffer (50 mM KH2PO4, 50 mM glycine

pH 9.0, 1% bentonite, 1% celite) was used to rub-inoculate leaves of one-week-old plants at two-

three leaf stage as described previously (Qi et al., 2008). Inoculated plants were covered (to

maintain humidity) and kept in the dark for 24 h. Subsequently, plants were transferred to diurnal

growth conditions and infection was determined by scoring for symptoms of chlorosis, necrosis,

and stunting observed in a typical infection. During the course of our study, we noticed that the

onset of the chlorosis symptoms on PMV/SPMV-infected Brachypodium varied in independent

infections performed at different times. The first visual chlorosis symptoms on the non-

inoculated leaves typically appear by 10 days-post-infection (dpi), but oftentimes appear earlier

at 7 dpi or were delayed till 14 dpi. This broad range in the onset of PMV/SPMV disease

symptoms could be due to inherent variation associated with the PMV/SPMV infectivity and/or

influence of environmental factors—factors that are not uncommon in similar pathosystems (Cui

et al., 2012). Nevertheless, to maintain consistency we advise maintaining similar growth

parameters particularly light-intensity, temperature, vernalization period etc., all of which

affected PMV/SPMV virulence (data not shown).



Mandadi, p. 26

Chlorophyll quantification

Chlorophyll was extracted, and measurements were performed as described previously

(Goldberg and Brakke, 1987; Bedell and Hangarter, 2004). Briefly, 100 mg of fresh plant tissue

was finely ground in liquid nitrogen and was mixed with 2 ml of 80% acetone. The acetone

extract was centrifuged at 13,000 g for 10 min at 4 °C. The supernatant was separated and the

pellet was re-suspended in 1 ml of 80% acetone followed by centrifugation and separation of

supernatant. The acetone extraction was repeated until the pellet was devoid of green

chlorophyll. Finally, all the supernatants were combined and chlorophyll absorbance was

measured using spectrophotometer at 663 (chlorophyll A) and 646 nm (chlorophyll B) and

converted to µg chlorophyll/mg fresh weight of leaf tissue (Lichtenthaler and Wellburn, 1983;

Goldberg and Brakke, 1987).

Protein extraction and immunoblot assays

Viral detection in infected plants was determined by immunoblot assays using rabbit

polyclonal antibodies against PMV CP and SPMV CP, as described previously (Scholthof,

1999). Briefly, plant samples were harvested and homogenized directly in 2X Laemmli SDS

sample buffer, as described previously (Martínez-García et al., 1999). The extracts were then

boiled for 10 min and centrifuged at 10,000 rpm for 5 min. The supernatants were

electrophoresed on SDS-PAGE gels (12.5%) and blotted onto nitrocellulose membranes

(Amersham). The blots were blocked in 5% milk followed by incubation with primary anti-PMV

CP (1:5000) or anti-SPMV CP (1:1000) serum and peroxidase-conjugated goat anti-rabbit IgG

secondary antibodies (Rockland) at 1:10,000 dilutions. Visualization of the proteins was

performed by using SuperSignal West Pico (Pierce) chemiluminescence detection reagents and

X-ray film.

RNA isolation, microarray and qRT-PCR analysis

Total RNA was isolated from shoots (inoculated and non-inoculated) of PMV and

PMV+SPMV co-infected plants at stage II, with only mild chlorosis symptoms at 7 dpi, and with

detectable PMV CP and SPMV CP (data not shown), along with mock-inoculated shoots, using

plant RNAeasy kit (Qiagen) following the manufacturer’s instructions. RNA from three

independent biological replicates was submitted to Nottingham Arabidopsis Stock Center



Mandadi, p. 27

(NASC) Affymetrix Service for labeling and hybridization. Standard Affymetrix

recommendations and quality controls were employed for microarray hybridization using a

custom-designed Affymetrix Brachypodium tiling array (BradiAR1b520742), consisting of

about 2.5 million probes representing approximately 27,000 genes, with greater than 95% of

exons and introns targeted by more than 5 probes. Mean probe size was about 42 bases and the

probes are spaced approximately 126 nucleotides apart. The gene predictions developed for this

array are based on the Brachypodium (accession Bd21) genome sequence, Illumina and EST-

based transcriptome analyses (The International Brachypodium Initiative, 2010) and are

accessible through Phytozome (v7.0, released on 8 April, 2011. For further information on the

array design please refer to Fox et al (Fox et al., 2010). A robust multichip average (RMA)-

sketch was employed for raw data normalization, background correction, and summarization of

the chip signals. Transcript-level sense-target probe sets were filtered out for further analysis.

Statistical significances among the normalized data sets was determined using Student’s t-test

and was corrected for false discovery rate (FDR) using the Benjamini and Hochberg method (q-

value <0.05) (Benjamini and Hochberg, 1995). Assignment of putative functions to the mis-

expressed genes was performed using a combined gene ontology (GO) using AgriGO resource

(Du et al., 2010), as well as, using Pfam (Punta et al., 2012) and PANTHER (Thomas et al.,

2003) annotations through Phytozome (Goodstein et al., 2012). The annotations were trimmed

for redundant GOs and multiple descriptions for similar biological processes and are summarized

in Figure 3B. Enriched GO terms within the PMV and PMV+SPMV co-infection were subjected

to singular enrichment analysis (SEA) against a pre-calculated Brachypodium background

genomic loci using the AgriGO tool (Du et al., 2010). Chi-square test and false discovery rate

(FDR) correction using the Hochberg method (FDR <0.05) was employed for determining

statistical significance. Significantly altered expression in specific functional classes of genes

were represented as heat maps, generated using the Multi-experiment Viewer (MeV) application

(Saeed et al., 2003). For identifying Arabidopsis orthologs of candidate Brachypodium genes, we

used the peptide homolog (PH) tool available through Phytozome (Goodstein et al., 2012). The

PH tool employs “all-against-all Smith-Waterman alignment” (Smith and Waterman, 1981),

which is considered superior to BLAST or FASTA based alignments for recovery of orthologous

peptides to a query sequence (Pearson, 1991; Shpaer et al., 1996). Reciprocal searches yielded

similar results. However, because cross-species comparisons of orthologous genes (monocots



Mandadi, p. 28

and dicots split ~150 million years ago (Chaw et al., 2004)) can be complicated, we took a

conservative approach and named only those Brachypodium genes after an Arabidopsis ortholog

(e.g., BdPR-1), only if they had unique and highest similarity scores. Others were assigned as “-

like” genes (e.g., PR-1-like). Further, for genes families such as WRKYs, protein kinases, ACO,

AOX, FAD, LOX, MKK, OPR and ERF proteins, multiple sequence alignment and phylogenetic

analyses was performed using the predicted protein sequences of Brachypodium genes and their

respective Arabidopsis orthologs retrieved from Phytozome were aligned in ClustalW2 (Larkin

et al., 2007). The resulting neighbor-joining trees were displayed using NJplot (Perrière and

Gouy, 1996) and/or Archeopterix (Han and Zmasek, 2009) display tools (Figure 5, 7 and S2A-

G). During the preparation of this manuscript Phtozome v8.0 was released (01/16/2012),

resulting in minor changes to a few gene loci. We manually screened for these changes and

incorporated them in our analysis as of 07/03/2012. Minimum Information About a Microarray

Experiment (MIAME)-compliant data was deposited in ArrayExpress (Parkinson et al., 2010)

with submission ID _______________.

Microarray data was further verified for reliability using (q) RT-PCR analysis of twenty

genes using the same RNA used for the microarray analysis. Although (q) RT-PCR analysis only

confirms the selected gene expression data, it is a commonly regarded independent laboratory-

based approach to corroborate microarrays, as microarrays are often prone to several procedural

and data-processing artifacts (Chuaqui et al.). For an unbiased representation of the microarray

data, we chose the twenty candidate genes in diverse functional categories with expression

changes ranging from 2 to 50-fold in PMV and PMV+SPMV infections, when compared to

mock-inoculated plants (Table S1). Further, temporal analysis of SA, JA, and ET responses was

performed using (q) RT-PCR analysis of candidate genes in the respective signaling pathways,

using plants at different stages of infection scored as: no symptoms (stage I), mild chlorosis

symptoms (stage II), or severe chlorosis and necrosis symptoms (stage III), as well as mock-

inoculated plants (Figure 6). For all (q) RT-PCR analysis, three independent biological

replicates consisting of three plant samples each was used for total RNA isolation using Trizol

reagent (Ambion), following the manufacturer’s instructions. Two µg of the total RNA was used

to make complementary DNA (cDNA) using SuperScript III first-strand cDNA synthesis kit

(Invitrogen), as described previously (Mandadi et al., 2009), with minor modifications. Gene

amplification by PCR was performed using Power SYBR Green Master-mix and the ABI Prism



Mandadi, p. 29

7500 sequence detection system (Applied Biosystems). Gene specific primers used for (q) RT-

PCR analysis were designed using QuantPrime (Arvidsson et al., 2008), and are listed in Table

S5 and S6. UBIQUITIN18 (Bradi4g00660) expression was used for normalization, and fold

changes of gene expression in the infected plants were calculated following the ΔΔCT method

(Livak and Schmittgen, 2001), and are relative to mock-inoculated plants set to 1. Student’s t-test

was used to determine statistical significances between the control and infected plants, and

Pearson correlation coefficient (r) and coefficient of determination (R2) were used to determine

statistical correlation between the microarray and the (q) RT-PCR data (Table S1).

ACKNOWLEDGMENTS

We thank Todd Mockler (Danforth Plant Science Center, St. Louis) for statistical analysis

of the microarray data and advice on gene functional annotations, and Joshua Yuan and Julia

Cope (Texas A&M University) for initial computer support. The generous gifts of

Brachypodium seed and practical advice from John Vogel, Jennifer Bragg, and Ludmilla Tyler

(USDA-ARS, Albany, CA), made it possible for us to develop Brachypodium for our virus

studies. We appreciate the helpful discussions of Herman Scholthof, Veria Alvarado, and

Christopher Lyons and their critical comments during this manuscript preparation. We thank

Jesse Pyle for help with growth and maintenance of the Brachypodium plants.



Mandadi, p. 30

Table I. Differential expression of putative Brachypodium genes involved in salicylic acid

signaling. The relative unlogged fold-change (≥1.5-fold) in gene expression of PMV and

PMV+SPMV infection compared to mock are indicated. Minus (-) represents down-regulated

fold-change. The gene descriptions were deduced from peptide ortholog analysis with

Arabidopsis protein sequences using Phytozome (Goodstein et al., 2012). The percentage

similarity of Brachypodium proteins with the orthologous Arabidopsis proteins are indicated in

parenthesis. Note that some Brachypodium genes with low percentage similarity to their

respective Arabidopsis orthologs are indicated as “-like”. For additional phylogenetic analyses,

please see Figures 5 and S2. The statistical significance of gene expression changes was

determined using Student’s t-test and was corrected for false discovery rate using the Benjamini

and Hochberg method (q-value <0.05).

Gene ID PMV PMV+ SPMV

Description

Bradi1g57580 42.53 21.90 PR-1-like (52%) Bradi1g57590 40.79 22.56 PR-1-like (52%) Bradi2g60490 10.23 9.47 BdPR-2 (51%) Bradi2g47210 3.76 3.32 BdPR-3 (71%) Bradi3g48230 2.39 2.07 PR-3-like (60%) Bradi4g14920 9.29 6.61 BdPR-4 (67%) Bradi1g13060 19.13 12.87 PR-5-like (54%) Bradi1g13070 26.34 19.88 PR-5-like (48%) Bradi1g67240 2.58 2.33 ICS1-like (19%) Bradi1g71530 3.36 1.83 BdAGD2 (73%) Bradi4g23367 -1.86 -2.18 PAD4-like (38%) Bradi3g32990 1.62 1.76 FMO1-like (32%) Bradi5g20547 28.99 32.88 BdAOX1A (90%) Bradi5g20540 22.16 23.93 BdAOX1A-like (70%) Bradi5g20557 6.01 6.38 BdAOX1B (86%) Bradi2g15877 20.59 23.90 WRKY53-like (42%) Bradi2g30695 4.96 5.28 WRKY70-like (12.4%) Bradi4g30360 3.67 2.96 WRKY18-like (43%)



Mandadi, p. 31

Table II. Differential expression of putative Brachypodium genes involved in jasmonic acid and

ethylene signaling. The relative unlogged fold-change (≥1.5-fold) in gene expression of PMV

and PMV+SPMV infection compared to mock are indicated. Minus (-) represents down-

regulated fold-change. The gene descriptions were deduced from peptide ortholog analysis with

Arabidopsis protein sequences using Phytozome (Goodstein et al., 2012). The percentage

similarity of Brachypodium proteins with the orthologous Arabidopsis proteins are indicated in

parenthesis. Note that some Brachypodium genes with low percentage similarity to their

respective Arabidopsis orthologs are indicated as “-like”. For additional phylogenetic analyses,

please see Figure S2. The statistical significance of gene expression changes was determined

using Student’s t-test and was corrected for false discovery rate using the Benjamini and

Hochberg method (q-value <0.05).


Description

Bradi1g05870 3.60 3.07 BdOPR3 (66%) Bradi2g35907 2.43 2.51 OPR3-like (52%) Bradi1g16580 -2.00 -3.03 BdFAD7 (71%) Bradi1g51330 -1.64 -1.89 VSP1-like (44%) Bradi3g01110 -1.64 -1.92 BdAOS (56%) Bradi3g30510 4.02 4.75 BdCYP89A6 (65%) Bradi3g07010 -1.96 -2.86 BdLOX2 (66%) Bradi3g07000 -2.56 -2.56 LOX2-like (64%) Bradi3g39980 6.30 7.68 LOX-like (62%) Bradi3g11260 1.60 1.74 BdMKK3 (64%) Bradi3g50490 -2.50 -2.70 BdERF-1 (42%) Bradi1g72450 -8.33 -7.69 ERF/RAP2.2-like (28%) Bradi5g25570 -4.76 -5.26 ERF3-like (28%) Bradi2g52370 -2.63 -2.44 ERF4-like (31%) Bradi1g75960 2.40 2.51 BdACO1 (82%)



Mandadi, p. 32

Table III. Differential expression of putative Brachypodium kinases altered in PMV and

PMV+SPMV infection. The relative unlogged fold-change (≥2-fold) in gene expression of PMV

and PMV+SPMV infection compared to mock are indicated. Minus (-) represents down-

regulated fold-change. The gene descriptions were deduced from Pfam and PANTHER

functional annotations obtained using the Phytozome (Goodstein et al., 2012). The statistical

significance of gene expression changes was determined using Student’s t-test and was corrected

for false discovery rate using the Benjamini and Hochberg method (q-value <0.05).


Description

Bradi4g02020 5.15 5.36 Leucine-rich repeat (LRR) protein kinase, receptor-like protein kinase (RLK) plant-type, subfamily LRR-VIII-2

Bradi1g32630 2.25 2.10 Protein kinase domain, RLK plant-type, subfamily LRR-VIII-2

Bradi4g11740 23.97 21.17 LRR N-terminal domain, RLK plant-type, subfamily LRR-XII

Bradi5g21857 2.91 3.02 LRR, Di-glucose binding within endoplasmic reticulum, RLK plant-type

Bradi4g02850 5.19 6.07 Wall-associated kinase (WAK), RLK plant-type, subfamily WAK/LRK10L-1

Bradi1g02210 6.88 8.33 Calcium-binding EGF domain, WAK, RLK plant-type

Bradi3g01157 2.66 2.55 Calcium-binding EGF domain, WAK, RLK plant-type

Bradi4g21990 4.05 4.53 Protein kinase domain, RLK plant-type, sub-family RLCK-OS2

Bradi1g75140 2.40 2.31 Protein kinase domain, RLK plant-type, subfamily RLCK-IV

Bradi4g38690 3.68 4.76 U-box domain, protein kinase domain, RLK plant-type, subfamily RLCK-IXb

Bradi4g37220 5.72 5.36 D-mannose binding lectin, S-locus glycoprotein, RLK plant-type, subfamily SD-1c

Bradi5g02980 3.51 2.82 D-mannose binding lectin, RLK plant-type, subfamily SD-2b

Bradi2g20687 2.37 3.00 D-mannose binding lectin, S-locus glycoprotein, RLK plant-type

Bradi4g24590 3.20 4.19 Protein kinase domain, RLK plant-type Bradi2g47510 -5.26 -7.14 MAPKK-related, subfamily MEKK_ste11_MAP3K Bradi4g30380 -3.23 -3.33 NAF domain, Calcium/Calmodulin-dependent

protein kinase, subfamily KIN1/SNF1/Nim1_like (CAMK_2)



Mandadi, p. 33

Bradi4g45310 -2.22 -2.78 AGC kinases, PAS fold, subfamily PVPK_like (AGC_8)_kin82y

Bradi1g57700 6.33 6.94 Protein kinase domain, RLK plant-type, subfamily L-LEC

Bradi1g23710 5.64 4.92 Legume lectin domain, RLK plant-type, subfamily L-LEC





Bradi1g08990 -2.38 -2.00 Protein kinase domain, subfamily L-LEC Bradi1g25647 2.32 2.56 Domain of unknown function DUF26,

RLK plant-type Bradi1g42397 -2.04 -2.13 Domain of unknown function DUF26,

RLK plant-type Bradi1g25717 2.54 2.26 Domain of unknown function DUF26,

RLK plant-type



Mandadi, p. 34

Table IV. Additively induced Brachypodium genes by SPMV. The relative unlogged fold-

change (≥2-fold) in gene expression of PMV and PMV+SPMV infection compared to mock are

indicated. Only gene expression changes that are statistically significant (p-value <0.05) between

PMV and PMV+SPMV infections are listed. The gene descriptions were deduced from Pfam and

PANTHER functional annotations obtained using the Phytozome (Goodstein et al., 2012). An

asterisk (*) represents genes that are also present in other tables and figures elsewhere in the

manuscript.


Description

Bradi3g31720* 117.20 164.83 Glutathione S-transferase, N-terminal domain Bradi2g49067 78.57 119.88 Unknown Bradi1g74970 73.33 96.13 Unknown Bradi3g16650 52.07 65.42 Unknown Bradi2g48160 47.15 61.90 Eukaryotic porin Bradi3g31727 40.47 53.87 Unknown Bradi1g06980 38.47 50.74 DnaJ domain Bradi2g44820 21.24 27.73 UDP-glucoronosyl and UDP-glucosyl transferase Bradi1g21921 16.93 22.52 Sulfotransferase 4B (ST4B) Bradi2g10857 12.29 20.97 Unknown Bradi1g59240 9.75 18.45 Unknown Bradi3g21270 10.61 15.96 Protein of unknown function (DUF1218) Bradi3g60470 6.61 11.70 Metallo-beta-lactamase superfamily Bradi3g28120 7.78 10.83 Unknown Bradi1g69140 7.64 10.25 RST domain of plant C-terminal Bradi5g26080 8.36 10.15 Dihydroorotate dehydrogenase Bradi3g09120 5.93 8.87 Peroxidase Bradi3g53227 6.25 8.17 Unknown Bradi2g48540 6.55 7.94 Unknown Bradi3g49330* 4.99 7.92 Ubiquitin family Bradi1g57657 5.45 7.01 Unknown Bradi2g18630 5.40 6.62 Eukaryotic porin Bradi3g38050 4.00 6.17 Carboxylesterase, alpha/beta hydrolase fold Bradi3g48590 4.47 5.97 Lipocalin/cytosolic fatty-acid binding protein family Bradi5g08520 4.21 5.93 K+ potassium transporter Bradi3g14140 3.91 5.05 Transmembrane amino acid transporter protein,

Tryptophan/tyrosine permease family Bradi2g14610 3.38 5.03 Holliday junction DNA helicase ruvB N-terminus,

ATPase family Bradi1g77680 3.39 4.83 Unknown



Mandadi, p. 35

Bradi1g00700 3.70 4.80 Sugar (and other) transporter, Major facilitator superfamily

Bradi4g38690* 3.68 4.76 Protein kinase domain, Universal stress protein family, U-box domain

Bradi3g22880 3.27 4.76 MatE protein, Multidrug resistance pump Bradi4g27850* 3.40 4.67 AP2 domain Bradi2g55892 3.48 4.59 Unknown Bradi2g44150* 2.74 4.30 Cytochrome P450 Bradi2g50630 3.55 4.29 NLI interacting factor-like phosphatase Bradi1g57690* 3.38 4.23 Legume lectin domain, Protein kinase domain Bradi4g24590* 3.20 4.19 Protein kinase domain Bradi3g21310 3.33 4.16 Miro-like protein, RAS family, EF hand associated Bradi1g11780 3.24 4.15 ABC1 family Bradi2g47020 3.01 4.05 Unknown Bradi2g44200* 2.48 3.96 Cytochrome P450 Bradi3g16450 3.24 3.92 SPFH domain /Band 7 family, Stomatin-like integral

membrane domain Bradi3g09620 2.60 3.91 Unknown Bradi3g52367 2.94 3.66 Unknown Bradi1g45470* 2.50 3.66 AP2 domain Bradi5g12530 2.92 3.59 SPFH domain /Band 7 family, Stomatin-like integral

membrane domain Bradi1g10840 2.62 3.53 UDP-glucoronosyl and UDP-glucosyl transferase Bradi1g26850 2.44 3.35 C2 domain, Ca2+-dependent phospholipid-binding

protein Bradi1g20995 2.48 3.31 Unknown Bradi5g07380 2.45 3.10 Cleavage site for pathogenic type III effector

avirulence factor Avr Bradi1g14350 2.49 3.09 MatE protein, Multidrug resistance pump Bradi2g20687* 2.37 3.00 D-mannose binding lectin, S-locus glycoprotein,

RLK plant-type Bradi3g52830 2.40 2.94 ATPase family associated with various cellular

activities (AAA) Bradi2g51592 2.29 2.90 Unknown Bradi5g02980* 3.51 2.82 D-mannose binding lectin, Protein kinase domain Bradi3g47580 2.14 2.62 Zinc finger, ZZ type, UBA/TS-N domain, Ubiquitin-

associated Bradi3g48690 2.02 2.52 Unknown



Mandadi, p. 36

Table V. Additively down-regulated Brachypodium genes by SPMV. The relative unlogged

fold-change (≥2-fold) in gene expression of PMV and PMV+SPMV infection compared to mock

are indicated. Only gene expression changes that are statistically significant (p-value <0.05)

between PMV and PMV+SPMV infections are listed. The gene descriptions were deduced from

Pfam and PANTHER functional annotations obtained using the Phytozome (Goodstein et al.,

2012). An asterisk (*) represents genes that are also present in other tables and figures elsewhere

in the manuscript.


Description

Bradi5g05110* -9.09 -33.33 Helix-loop-helix DNA-binding domain Bradi4g42540* -8.33 -14.29 Cytochrome P450 Bradi4g36601 -3.70 -12.50 Aquaporin transporter Bradi2g05510* -3.45 -6.67 WRKY DNA-binding domain Bradi5g17500 -3.33 -6.25 NUDIX domain, phosphohydrolase Bradi2g55050 -4.00 -5.88 Multicopper oxidase Bradi1g12130* -2.63 -5.26 Chlorophyll A/B binding protein Bradi2g05832 -2.50 -4.55 Unknown Bradi2g20380* -2.78 -4.55 Photosystem II reaction centre W protein (PsbW) Bradi2g54430 -2.86 -4.35 Unknown Bradi1g42670* -2.63 -4.35 Chlorophyll A/B binding protein Bradi3g32200 -2.94 -4.17 Unknown Bradi2g56030 -2.56 -4.00 Fructose-1-6-bisphosphatase Bradi3g37680 -2.56 -3.85 Cupin domain Bradi4g20500 -2.38 -3.85 GUN4-like, magnesium-protoporphyrin IX

synthesis Bradi2g62520 -2.78 -3.70 Major intrinsic protein, Aquaporin Bradi1g68210 -2.63 -3.70 Senescence-associated protein Bradi3g07190* -2.56 -3.70 Chlorophyll A/B binding protein Bradi5g22290 -2.17 -3.57 Aminomethyltransferase folate-binding domain Bradi3g10770 -2.56 -3.33 Unknown Bradi3g04930 -2.17 -3.23 Ribosomal RNA adenine dimethylase, ubiE/COQ5

methyltransferase family Bradi2g26280 -2.50 -3.23 Unknown Bradi3g60920 -2.00 -3.13 NAD dependent epimerase/dehydratase family,

Male sterility protein Bradi1g55720 -2.08 -2.94 Leucine-rich repeat serine/threonine phosphatase Bradi5g01737 -2.13 -2.86 Unknown



Mandadi, p. 37

Bradi3g13970* -2.13 -2.86 Photosystem II protein Y (PsbY) Bradi4g45310* -2.22 -2.78 Protein kinase domain Bradi1g24870* -2.00 -2.63 Chlorophyll A/B binding protein Bradi5g17480* -2.04 -2.63 AP2 domain



Mandadi, p. 38

Table VI. Unique Brachypodium nuclear transcription and splicing factors altered by SPMV.

Differential expression of putative splicing factors and transcription factors uniquely altered in

PMV+SPMV infection. The fold-changes in gene expression in PMV and PMV+SPMV

infections compared to mock are shown. Changes in gene expression in PMV+SPMV infection

as determined by microarray and (q) RT-PCR analysis strongly correlate to each other (Pearson

correlation coefficients (r) = 0.95 and coefficient of determination (R2) = 0.92) and are

statistically significant compared to mock and PMV infections. Changes in gene expression in

PMV infection and mock are insignificant. The statistical significance of (q) RT-PCR changes in

gene expression was determined using Student’s t-test (p-value <0.05). For the microarray data,

False Discovery Rate correction using the Benjamini and Hochberg method (q-value <0.05) was

used. Primers used for this study are listed in Table S6.The gene descriptions were deduced from

Pfam and PANTHER functional annotations obtained using the Phytozome (Goodstein et al.,

2012).


PMV PMV+ SPMV

Description

Microarray (q) RT-PCR Bradi2g09850 1.11 1.58 0.90 1.28 Pre-mRNA splicing factor PRP21 like

protein, splicing factor 3A subunit 1 Bradi5g07850 1.11 2.09 1.30 1.84 Splicing factor 1/branch point binding

protein (RRM superfamily), Zinc knuckle domain

Bradi3g06562 1.16 1.55 1.00 1.34 AP2 domain Bradi4g34700 1.02 1.63 1.10 1.50 Unknown



Mandadi, p. 39

FIGURE LEGENDS

Figure 1. SPMV exacerbates disease symptoms and enhances PMV accumulation in

Brachypodium distachyon. (A, B). Typical disease symptoms and gross effects of PMV alone

and PMV+SPMV co-infection at 42 days-post-inoculation (dpi). PMV alone infection causes

mild chlorosis, necrosis and stunting of roots and shoots, while PMV+SPMV co-infection causes

severe stunting, chlorosis, and reduced seed-set. The inset image in “A” shows the severe

chlorosis associated with co-infection with SPMV. The progression of PMV and PMV+SPMV

infection can be classified into: (C) stage I with no visible chlorosis on leaves, stage II with mild

chlorosis on leaves, and stage III with prominent chlorosis and necrosis on leaves, stunting and

reduced seed-set. Immunoblot analysis of capsid protein accumulation at infection stages I, II

and III with (D) PMV alone or (E) PMV+SPMV infection. The earlier detection of PMV CP in

the mixed infections (E) is associated with SPMV enhancement of either PMV replication or

movement, which is a component of the working definition of the SPMV-associated synergism

(Scholthof, 1999). The asterisks (*) represent lower molecular-weight CPs, possibly produced

from leaky scanning (Omarov et al., 2005) or post-translational modifications. The Ponceau S

stained nitrocellulose membranes serve to indicate approximately equal protein loading among

the sample lanes.

Figure 2. Brachypodium distachyon infections with PMV+SPMV compared to mock-

inoculated plants. Typical outcomes of PMV+SPMV infection result in (A) leaf chlorosis and

necrosis (indicated by arrows), (B) stunting, (C) reduced chlorophyll A and chlorophyll B (Chl A

and Chl B) accumulation by stage III, (D) decreased fresh weight (FW) and dry weight (DW),

(E) shorter internodes, (F) smaller leaves, but no significant effect on tiller number (G). Asterisks

in the graph represent significant differences compared to mock controls (p-value <0.01, student

t-test). (H) Immunoblot assays of PMV CP and SPMV CP accumulation in PMV+SPMV

infected roots and shoots at stage III. The asterisk (*) represents a lower molecular-weight

SPMV CP (see legend of Figure 1 and (Omarov et al., 2005)). The Coomassie Brilliant Blue R-

stained gel reflects approximate equal sample loading among the mock- and virus-inoculated

sample lanes.



Mandadi, p. 40

Figure 3. PMV and SPMV alter genes in diverse functional categories. Triplicate samples of

Brachypodium (Bd21-3) plants infected with PMV or PMV+SPMV, or mock-inoculated at stage

II (mild chlorosis symptoms) were analyzed using microarrays. Significant changes in gene

expression relative to mock were determined using the Benjamini and Hochberg (BH) method by

correcting for false discovery rate (q-value <0.05). (A) The Venn diagrams represent the number

of genes up-regulated and down-regulated in response to PMV and PMV+SPMV infection. (B)

Summary of the biological processes overrepresented in PMV- and PMV+SPMV-infections as

deduced using AgriGO (Du et al., 2010), Pfam and PANTHER functional annotations (Thomas

et al., 2003; Punta et al., 2012). A detailed list of the GO assignments and SEA analysis is in

Table S2.

Figure 4. Pseudo-colored heat map representation of Brachypodium gene expression in

PMV alone and PMV+SPMV infection. The color bars on the top indicate logarithmic

expression values (log2) from the microarray; green for lower expression and red for higher

expression. (A) The up-regulated glutathione S-transferase proteins, chaperone/heat shock

proteins (HSPs), cytochrome P450 family proteins, and several genes implicated in protein

synthesis are indicated. (B) The down-regulated photosynthesis-related genes are indicated (see

also Figure 2C for effects on chlorophyll content). Functional classification of the different genes

was deduced using AgriGO (Du et al., 2010), Pfam, and PANTHER annotations (Thomas et al.,

2003; Punta et al., 2012). Note that genes with multiple alternatively spliced transcripts occur

more than once, and certain alternatively spliced transcripts exhibit differential expression

kinetics.

Figure 5. Sequence analysis of PMV and SPMV modulated WRKY transcription factors in

Brachypodium. (A) Multiple sequence alignment of WRKY-domain sequence of WRKY

factors mis-expressed in PMV and SPMV infection. The consensus symbols are indicated below

the alignments with identically conserved residues indicated by black shading and an asterisk (*).

Amino acids with ≥ 50% identity are shaded grey and marked with a period (•). The conserved

WRKY and zinc-finger motifs are underlined and in red and blue, respectively. (B) The

phylogenetic relationship of Brachypodium WRKY factors mis-expressed in PMV and

PMV+SPMV infections with orthologous Arabidopsis WRKY factors. The protein sequences



Mandadi, p. 41

were retrieved from Phytozome (Goodstein et al., 2012), aligned using ClustalX2 (Larkin et al.,

2007), and displayed as an unrooted tree using Archeopteryx (Han and Zmasek, 2009). The

branch length at bottom right of the phylogram indicates genetic distances. Down-regulated

genes are identified by black triangles. The grouping of WRKY factors into Group Ic, IIa, IIc

and III was based on the classification described previously (Rushton et al., 2010).

Figure 6. Temporal expression analysis of SA, JA and ET components during PMV and

SPMV disease progression. Brachypodium plants infected with PMV, PMV+SPMV, or mock-

inoculated sampled at stages I, II and III (see Figure 1C for description of stages) to determine

the expression of putative genes in SA responses (A) PR-1-like, (B) PR-3-like, (C) PR-5-like,

(D) PAD4-like, and (E) AOX1A; JA responses (F) LOX2, (G) AOS and (H) OPR3; and, ET

responses (I) ERF-1 and (J) ERF3-like using (q) RT-PCR analysis. Transcript level of

UBIQUITIN18 (UBC18) was used to normalize the (q) RT-PCR data. Expression values plotted

are average of three biological replicates. Error bars represent standard deviation among the

replicates. Significant changes in gene expression was determined using Student’s t-test (p-value

<0.05). Note that the expression scales for each gene panel are unique and are relative to the

mock-inoculated control plants at stage I. Primers used are listed in Table S7.

Figure 7. Phylogenetic classification of Brachypodium serine-threonine kinases altered in

PMV and PMV+SPMV infection. The sequences were retrieved from Phytozome (Goodstein

et al., 2012), aligned using ClustalX2 (Larkin et al., 2007), and displayed as an unrooted tree

using Archeopteryx (Han and Zmasek, 2009). The functional domains and sub-families of the

kinases were deduced using predicted Pfam and PANTHER annotations (Thomas et al., 2003;

Punta et al., 2012) using Phytozome (Goodstein et al., 2012). The kinases were grouped into

subfamilies: LRR-VIII-2, LRR-XII , WAK/LRK10L-1, RLCK-OS2, RLCK-IV, RLCK-IXb, SD-

1c , SD-2b, MEKK_ste11_MAP3K , KIN1/SNF1/Nim1_like (CAMK_2), PVPK_like_kin82y

(AGC_8), L-LEC, and DUF26, following a classification previously described (Shiu et al.,

2004). Down-regulated genes are identified by black triangles. Note that among the down-

regulated genes, Bradi2g47510, Bradi4g30380, and Bradi4g45310 cluster together, suggesting a

transcriptional co-regulation of these phylogenetically-related kinases.



Mandadi, p. 42

Figure 8. Proposed model for the host:pathogen interactions induced by PMV alone or the

PMV+SPMV synergism in Brachypodium. Briefly, the PMV replicase-associated proteins

(P48, P112), movement-associated proteins (P8, P6.6, P15 ), PMV capsid protein (CP) and

SPMV capsid protein (SPCP) cooperatively function in translation, replication, encapsidation,

and movement of PMV and SPMV in a systemic infection of Brachypodium and other grasses in

the Poaceae. PMV and SPMV move cell-to-cell primarily via plasmodesmata (up-down arrow)

as viral ribonucleoprotein (vRNP) complexes, probably with assistance from PMV P8, SPCP

(which are also associated with the host cell-membrane), P6.6, and P15 (Turina et al., 2000; Qi et

al., 2008). Blue and orange beads on a string represent PMV and SPMV vRNPs, respectively.

Both PMV virions (blue hexagon) and SPMV virions (orange hexagon) assemble in the cytosol

of an infected leaf and are probably introduced into new cells through cell damage (lightning

bolt). An as of yet unidentified PMV- or SPMV-associated viral component (RNA and/or

proteins indicated by “?”) elicits host defense responses activating expression of multiple

transmembrane receptor-like kinases (TM-RLK) and cytoplasmic receptor-like kinases (Cyto-

RLK). PMV and PMV+SPMV infection also triggers expression of salicylic acid (SA)-,

jasmonic acid (JA)-, and ethylene components, possibly as a host defense response (the red and

green text indicates up- and down-regulated genes, respectively). SPCP also localizes to the host

nucleus and nucleolus (Qi et al., 2008) thereby uniquely modulating expression of putative

splicing factors (SF1 and SF3A) and an AP2 factor and may affect alternative splicing of certain

host mRNAs. The annotation of Brachypodium genes is based on orthologous Arabidopsis

proteins. Further outcomes of the Brachypodium transcriptome changes in response to PMV and

PMV+SPMV infections are explained in the Discussion. The PMV and SPMV genomes are

shown in Figure S1.

Figure S1. Panicum mosaic virus (PMV) and satellite panicum mosaic virus (SPMV)

genome maps and infection strategy. (A) PMV, a single-stranded positive-sense (ss) RNA

virus encodes the 48 kDa (P48) and 112 kDa (P112) replicase proteins from a 4,326-nt genomic

RNA (Batten et al., 2006b). The P8, P6.6, P15, and the 26-kDa capsid protein (CP; P26) are

encoded on a single subgenomic RNA and contribute to virus movement (Turina et al., 1998;

Turina et al., 2000). The 3'-end of the PMV RNA has a translational enhancer sequence that



Mandadi, p. 43

assists with cap-independent translation (Batten et al., 2006a). PMV assembles into T=3, 30 nm

virions from the P26 CP. SPMV is an 824-nt ssRNA encoding a 17-kDa CP (SPMV CP) to form

T=1, 16 nm virions (Ban et al., 1995; Ban and McPherson, 1995). (B) The cartoon shows the

outcomes from PMV alone, SPMV alone, and PMV+SPMV infections. PMV can replicate and

move in host plants; SPMV alone is not infectious; and, the mixed infection of PMV+SPMV

results in the replication and movement of both viral RNAs. The mixed infection is associated

with a more severe infection phenotype and increased titer of PMV, compared to PMV alone

infections.

Figure S2. Phylogenetic analysis of putative SA, JA and ET components altered in PMV

and PMV+SPMV infection. The predicted amino-acid sequences of Brachypodium (A) ACO,

(B) AOX, (C) ERF, (D) FAD, (E) LOX, (F) MKK and (G) OPR proteins mis-expressed in PMV

and PMV+SPMV infection (Tables I and II; indicated by an asterisk (*) in the phylogram) and

their corresponding Arabidopsis orthologs, were aligned using ClustalX2 (Larkin et al., 2007),

and the resulting neighbor-joining tree is displayed using N-J plot (Perrière and Gouy, 1996).

The branch length at top right end of the phylogram indicates genetic distances. Bootstrap values

from 1000 replicates are indicated at the branch nodes. Arabidopsis genes closest to the

Brachypodium genes within the same cluster are indicated by double asterisks (**).



Mandadi, p. 44

LITERATURE CITED

Afzal AJ, Wood AJ, Lightfoot DA (2008) Plant receptor-like serine threonine kinases: roles in signaling and plant defense. Mol Plant Microbe Interact 21: 507-517

Alamillo JM, Saénz P, García JA (2006) Salicylic acid-mediated and RNA-silencing defense mechanisms cooperate in the restriction of systemic spread of plum pox virus in tobacco. Plant J 48: 217-227

Alvarado V, Scholthof HB (2009) Plant responses against invasive nucleic acids: RNA silencing and its suppression by plant viral pathogens. Sem Cell Dev Biol 20: 1032-1040

Alvarado VY, Scholthof HB (2012) AGO2: a new Argonaute compromising plant virus accumulation. Front Plant Sci 2: 112

An C, Mou Z (2011) Salicylic acid and its function in plant immunity. J Integ Plant Biol 53: 412-428

Arvidsson S, Kwasniewski M, Riano-Pachon D, Mueller-Roeber B (2008) QuantPrime-a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinformatics 9: 465

Ascencio-Ibáñez JT, Sozzani R, Lee T-J, Chu T-M, Wolfinger RD, Cella R, Hanley-Bowdoin L (2008) Global analysis of Arabidopsis gene expression uncovers a complex array of changes impacting pathogen response and cell cycle during geminivirus infection. Plant Physiol 148: 436-454

Ayliffe MA, Steinau M, Park RF, Rooke L, Pacheco MG, Hulbert SH, Trick HN, Pryor AJ (2004) Aberrant mRNA processing of the maize Rp1-D rust resistance gene in wheat and barley. Mol Plant Microbe Interact 17: 853-864

Baebler Š, Stare K, Kovač M, Blejec A, Prezelj N, Stare T, Kogovšek P, Pompe-Novak M, Rosahl S, Ravnikar M, Gruden K (2011) Dynamics of responses in compatible potato-Potato virus Y interaction are modulated by salicylic acid. PLoS One 6: e29009

Ban N, Larson SB, McPherson A (1995) Structural comparison of plant satellite viruses. Virology 214: 571-583

Ban N, McPherson A (1995) The structure of satellite panicum mosaic virus at 1.9 Å resolution. Nature Struct. Biol. 2: 882-890

Batten JS, Desvoyes B, Yamamura Y, Scholthof K-BG (2006a) A translational enhancer element on the 3'-proximal end of the Panicum mosaic virus genome. FEBS Lett. 580: 2591-2597

Batten JS, Turina M, Scholthof K-BG (2006b) Panicovirus accumulation is governed by two membrane-associated proteins with a newly identified conserved motif that contributes to pathogenicity. Virol. J. 3: 12

Bedell V, Hangarter R (2004) Characterization of the Arabidopsis seedling plastid mutant spd2. BIOS 75: 65-73

Bell E, Creelman RA, Mullet JE (1995) A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis. Proc Natl Acad Sci USA 92: 8675-8679

Bendahmane A, Kanyuka K, Baulcombe DC (1999) The Rx gene from potato controls separate virus resistance and cell death responses. Plant Cell 11: 781-792

Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: A practical and powerful approach to multiple testing. J Royal Stat Soc Series B 57: 289-300



Mandadi, p. 45

Bevan MW, Garvin DF, Vogel JP (2010) Brachypodium distachyon genomics for sustainable food and fuel production. Curr. Opin. Biotechnol. 21: 211-217

Bleecker AB, Kende H (2000) Ethylene: A gaseous signal molecule in plants. Annu Rev Cell Dev Biol 16: 1-18

Brkljacic J, Grotewold E, Scholl R, Mockler T, Garvin DF, Vain P, Brutnell T, Sibout R, Bevan M, Budak H, Caicedo AL, Gao C, Gu y, Hazen SP, Holt BF, Hong S-Y, Jordan M, Manzaneda AJ, Mitchell-Olds T, Mochida K, Mur LAJ, Park C-M, Sedbrook J, Watt M, Zheng SJ, Vogel J (2011) Brachypodium as a model for the grasses: Today and the future. Plant Physiol 157: 3-13

Brutus A, Sicilia F, Macone A, Cervone F, De Lorenzo G (2010) A domain swap approach reveals a role of the plant wall-associated kinase 1 (WAK1) as a receptor of oligogalacturonides. Proc Natl Acad Sci USA 107: 9452-9457

Buzen FG, Niblett CL, Hooper GR, Hubbard J, Newman MA (1984) Further characterization of Panicum mosaic virus and its associated satellite virus. Phytopathology 74: 313-318

Cabrera O, Scholthof K-BG (1999) The complex viral etiology of St. Augustine decline. Plant Dis. 83: 902-904

Cao S, Siriwardana C, Kumimoto R, Holt BF (2011) Construction of high quality Gateway entry libraries and their application to yeast two-hybrid for the monocot model plant Brachypodium distachyon. BMC Biotechnol 11: 53

Carr JP, Lewsey MG, Palukaitis P (2010) Signaling in induced resistance. Adv Virus Res 76: 57-121

Chaw S-M, Chang C-C, Chen H-L, Li W-H (2004) Dating the monocot-dicot divergence and the origin of core eudicots using whole chloroplast genomes. J Mol Evol 58: 424-441

Chen X, Chern M, Canlas PE, Jiang C, Ruan D, Cao P, Ronald PC (2010) A conserved threonine residue in the juxtamembrane domain of the Xa21 pattern recognition receptor is critical for kinase autophosphorylation and Xa21-mediated immunity. J Biol Chem 285: 10454-10463

Chuaqui RF, Bonner RF, Best CJM, Gillespie JW, Flaig MJ, Hewitt SM, Phillips JL, Krizman DB, Tangrea MA, Ahram M, Linehan WM, Knezevic V, Emmert-Buck MR (2002) Post-analysis follow-up and validation of microarray experiments. Nat Genet 32: S509-514

Cui Y, Lee MY, Huo N, Bragg J, Yan L, Yuan C, Li C, Holditch SJ, Xie J, Luo M-C, Li D, Yu J, Martin J, Schackwitz W, Gu YQ, Vogel JP, Jackson AO, Liu Z, Garvin DF (2012) Fine mapping of the Bsr1 Barley stripe mosaic virus resistance gene in the model grass Brachypodium distachyon. PLoS ONE 7: e38333

Dalmay T, Horsefield R, Braunstein TH, Baulcombe DC (2001) SDE3 encodes an RNA helicase required for post-transcriptional gene silencing in Arabidopsis. EMBO J 20: 2069-2077

Damirdagh IS, Ross AF (1967) A marked synergistic interaction of potato viruses X and Y in inoculated leaves of tobacco. Virology 31: 296-307

Dardick C, Ronald P (2006) Plant and animal pathogen recognition receptors signal through non-RD kinases. PLoS Pathog 2: e2

Desvoyes B, Scholthof K-BG (2000) RNA:protein interactions associated with satellites of Panicum mosaic virus. FEBS Lett 485: 25-28

Dinesh-Kumar SP, Baker BJ (2000) Alternatively spliced N resistance gene transcripts: Their possible role in Tobacco mosaic virus resistance. Proc Natl Acad Sci USA 97: 1908-1913



Mandadi, p. 46

Dodds PN, Rathjen JP (2010) Plant immunity: towards an integrated view of plant–pathogen interactions. Nat Rev Genet 11: 539-548

Dong JG, Fernandez-Maculet JC, Yang SF (1992) Purification and characterization of 1-aminocyclopropane-1-carboxylate oxidase from apple fruit. Proc Natl Acad Sci USA 89: 9789-9793

Draper J, Mur LA, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge AP (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol. 127: 1539-1555

Du Z, Zhou X, Ling Y, Zhang Z, Su Z (2010) agriGO: a GO analysis toolkit for the agricultural community. Nucleic Acids Res 38: W64-W70

Dunoyer P, Voinnet O (2005) The complex interplay between plant viruses and host RNA-silencing pathways. Curr Opin Plant Biol 8: 415-423

Elena SF, Carrera J, Rodrigo G (2011) A systems biology approach to the evolution of plant-virus interactions. Curr Opin Plant Biol 14: 372-377

Everett AL, Scholthof HB, Scholthof K-BG (2010) Satellite panicum mosaic virus coat protein enhances the performance of plant virus gene vectors. Virology 396: 37-46

Fernandez-Calvino L, Faulkner C, Walshaw J, Saalbach G, Bayer E, Benitez-Alfonso Y, Maule A (2011) Arabidopsis plasmodesmal proteome. PLoS One 6: e18880

Fox SE, Priest HD, Bryant DW, Wilhelm LJ, Michael TP, Mockler TC (2010) Universal genome array and transcriptome atlas for Brachypodium distachyon. Abstract# W095 In Plant & Animal Genomes XVIII Conference. San Diego, CA (http://www.intlpag.org/18/abstracts/W13_PAGXVIII_095.html

García-Marcos A, Pacheco R, Martiáñez J, González-Jara P, Díaz-Ruíz JR, Tenllado F (2009) Transcriptional changes and oxidative stress associated with the synergistic interaction between Potato virus X and Potato virus Y and their relationship with symptom expression. Mol Plant Microbe Interact 22: 1431-1444

Garvin DF, McKenzie N, Vogel JP, Mockler TC, Blankenheim ZJ, Wright J, Cheema JJ, Dicks J, Huo N, Hayden DM, Gu Y, Tobias C, Chang JH, Chu A, Trick M, Michael TP, Bevan MW, Snape JW (2010) An SSR-based genetic linkage map of the model grass Brachypodium distachyon. Genome 53: 1-13

Goldberg K-B, Brakke MK (1987) Concentration of Maize chlorotic mottle virus increased in mixed infections with Maize dwarf mosaic virus, strain B. Phytopathology 77: 162-167

Gómez-Gómez L, Boller T (2002) Flagellin perception: a paradigm for innate immunity. Trends Plant Sci 7: 251-256

González-Jara P, Tenllado F, Martínez-García B, Atencio FA, Barajas D, Vargas M, DíAz-Ruiz J, Díaz-Ruíz JR (2004) Host-dependent differences during synergistic infection by Potyviruses with Potato virus X. Mol Plant Pathol 5: 29-35

Goodman RM, Ross AF (1974) Enhancement of Potato virus X synthesis in doubly infected tobacco occurs in doubly infected cells. Virology 58: 16-24

Goodstein DM, Shu S, Howson R, Neupane R, Hayes RD, Fazo J, Mitros T, Dirks W, Hellsten U, Putnam N, Rokhsar DS (2012) Phytozome: a comparative platform for green plant genomics. Nucleic Acids Res 40: D1178-D1186

Han M, Zmasek C (2009) phyloXML: XML for evolutionary biology and comparative genomics. BMC Bioinformatics 10: 356

Hanssen IM, Peter van Esse H, Ballester A-R, Hogewoning SW, Parra NO, Paeleman A, Lievens B, Bovy AG, Thomma BPHJ (2011) Differential tomato transcriptomic



Mandadi, p. 47

responses induced by Pepino mosaic virus isolates with differential aggressiveness. Plant Physiol 156: 301-318

Heil M, Ton J (2008) Long-distance signalling in plant defence. Trends Plant Sci 13: 264-272 Higuchi M, Pischke MS, Mähönen AP, Miyawaki K, Hashimoto Y, Seki M, Kobayashi M,

Shinozaki K, Kato T, Tabata S, Helariutta Y, Sussman MR, Kakimoto T (2004) In planta functions of the Arabidopsis cytokinin receptor family. Proc Natl Acad Sci USA 101: 8821-8826

Hu C-C, Hsu Y-H, Lin N-S (2009) Satellite RNAs and satellite viruses of plants. Viruses 1: 1325-1350

Huang Z, Yeakley JM, Garcia EW, Holdridge JD, Fan J-B, Whitham SA (2005) Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus interactions. Plant Physiol 137: 1147-1159

Huo N, Gu YQ, Lazo GR, Vogel JP, Coleman-Derr D, Luo MC, Thilmony R, Garvin DF, Anderson OD (2006) Construction and characterization of two BAC libraries from Brachypodium distachyon, a new model for grass genomics. Genome 49: 1099-1108

The International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463: 763-768

Ishibashi K, Masuda K, Naito S, Meshi T, Ishikawa M (2007) An inhibitor of viral RNA replication is encoded by a plant resistance gene. Proc Natl Acad Sci USA 104: 13833-13838

Jakubiec A, Yang SW, Chua N-H (2012) Arabidopsis DRB4 protein in antiviral defense against Turnip yellow mosaic virus infection. Plant J 69: 14-25

Jensen AB, Raventos D, Mundy J (2002) Fusion genetic analysis of jasmonate-signalling mutants in Arabidopsis. Plant J 29: 595-606

Jia M-A, Li Y, Lei L, Di D, Miao H, Fan Z (2012) Alteration of gene expression profile in maize infected with a double-stranded RNA fijivirus associated with symptom development. Mol Plant Pathol 13: 251-262

Jo Y, Cho W, Rim Y, Moon J, Chen X-Y, Chu H, Kim C, Park Z-Y, Lucas W, Kim J-Y (2011) Plasmodesmal receptor-like kinases identified through analysis of rice cell wall extracted proteins. Protoplasma 248: 191-203

Jordan T, Schornack S, Lahaye T (2002) Alternative splicing of transcripts encoding Toll-like plant resistance proteins – what's the functional relevance to innate immunity? Trends Plant Sci 7: 392-398

Kachroo P, Chandra‐Shekara AC, Klessig DF (2006) Plant signal transduction and defense against viral pathogens. Adv Virus Res 66: 161-191

Kachroo P, Yoshioka K, Shah J, Dooner HK, Klessig DF (2000) Resistance to Turnip crinkle virus in Arabidopsis is regulated by two host genes and is salicylic acid dependent but NPR1, ethylene, and jasmonate independent. Plant Cell 12: 677-690

Kim SH, Ryabov EV, Kalinina NO, Rakitina DV, Gillespie T, MacFarlane S, Haupt S, Brown JW, Taliansky M (2007) Cajal bodies and the nucleolus are required for a plant virus systemic infection. EMBO J 26: 2169-2179

Kohm BA, Goulden MG, Gilbert JE, Kavanagh TA, Baulcombe DC (1993) A Potato virus X resistance gene mediates an induced, nonspecific resistance in protoplasts. Plant Cell 5: 913-920



Mandadi, p. 48

Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG (2007) Clustal W and Clustal X version 2.0. Bioinformatics 23: 2947-2948

Latham JR, Wilson AK (2008) Transcomplementation and synergism in plants: implications for viral transgenes? Mol Plant Pathol 9: 85-103

Lennon AM, Neuenschwander UH, Ribas-Carbo M, Giles L, Ryals JA, Siedow JN (1997) The effects of salicylic acid and Tobacco mosaic virus infection on the alternative oxidase of tobacco. Plant Physiol 115: 783-791

Li J, Chory J (1997) A putative leucine-rich repeat receptor kinase involved in brassinosteroid signal transduction. Cell 90: 929-938

Lichtenthaler HK, Wellburn AR (1983) Determinations of total carotenoids and chlorophylls A and B of leaf extracts in different solvents. Biochem Soc Trans 11: 591

Lim PO, Kim HJ, Gil Nam H (2007) Leaf senescence. Annu Rev Plant Biol 58: 115-136 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time

quantitative PCR and the 2-ΔΔCT method. Methods 25: 402-408 Lorenzo O, Piqueras R, Sánchez-Serrano JJ, Solano R (2003) ETHYLENE RESPONSE

FACTOR1 integrates signals from ethylene and jasmonate pathways in plant defense. Plant Cell 15: 165-178

Mandadi KK, Misra A, Ren S, McKnight TD (2009) BT2, a BTB protein, mediates multiple responses to nutrients, stresses, and hormones in Arabidopsis. Plant Physiol 150: 1930-1939

Martínez-García JF, Monte E, Quail PH (1999) A simple, rapid and quantitative method for preparing Arabidopsis protein extracts for immunoblot analysis. Plant J 20: 251-257

Masuta C, Zuidema D, Hunter BG, Heaton LA, Sopher DS, Jackson AO (1987) Analysis of the genome of satellite panicum mosaic virus. Virology 159: 329-338

Moore JW, Loake GJ, Spoel SH (2011) Transcription dynamics in plant immunity. Plant Cell 23: 2809-2820

Mur LAJ, Kenton P, Atzorn R, Miersch O, Wasternack C (2006) The outcomes of concentration-specific interactions between salicylate and jasmonate signaling include synergy, antagonism, and oxidative stress leading to cell death. Plant Physiol 140: 249-262

Murphy AM, Chivasa S, Singh DP, Carr JP (1999) Salicylic acid-induced resistance to viruses and other pathogens: a parting of the ways? Trends Plant Sci 4: 155-160

Niblett CL, Paulsen AQ (1975) Purification and further characterization of Panicum mosaic virus. Phytopathology 65: 1157-1160

Omarov RT, Qi D, Scholthof K-BG (2005) The capsid protein of satellite panicum mosaic virus contributes to systemic invasion and interacts with its helper virus. J Virol 79: 9756-9764

Pacheco R, García-Marcos A, Barajas D, Martiáñez J, Tenllado F (2012) PVX–potyvirus synergistic infections differentially alter microRNA accumulation in Nicotiana benthamiana. Virus Res 165: 231-235

Padmanabhan MS, Dinesh-Kumar SP (2010) All hands on deck—the role of chloroplasts, endoplasmic reticulum, and the nucleus in driving plant innate immunity. Mol Plant Microbe Interact 23: 1368-1380

Pallas V, García JA (2011) How do plant viruses induce disease? Interactions and interference with host components. J Gen Virol 92: 2691-2705



Mandadi, p. 49

Parkinson H, Sarkans U, Kolesnikov N, Abeygunawardena N, Burdett T, Dylag M, Emam I, Farne A, Hastings E, Holloway E, Kurbatova N, Lukk M, Malone J, Mani R, Pilicheva E, Rustici G, Sharma A, Williams E, Adamusiak T, Brandizi M, Sklyar N, Brazma A (2010) ArrayExpress update—an archive of microarray and high-throughput sequencing-based functional genomics experiments. Nucleic Acids Res 39: D1002-1004

Pearson WR (1991) Searching protein sequence libraries: Comparison of the sensitivity and selectivity of the Smith-Waterman and FASTA algorithms. Genomics 11: 635-650

Perrière G, Gouy M (1996) WWW-query: An on-line retrieval system for biological sequence banks. Biochimie 78: 364-369

Pieterse CMJ, Leon-Reyes A, Van der Ent S, Van Wees SCM (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5: 308-316

Piron F, Nicolaï M, Minoïa S, Piednoir E, Moretti A, Salgues A, Zamir D, Caranta C, Bendahmane A (2010) An induced mutation in tomato eIF4E leads to immunity to two potyviruses. PLoS One 5: e11313

Postnikova O, Nemchinov L (2012) Comparative analysis of microarray data in Arabidopsis transcriptome during compatible interactions with plant viruses. Virology J 9: 101

Powell AM (1994) Grasses of the Trans-Pecos and adjacent areas. University of Texas Press, Austin

Pruss G, Ge X, Shi XM, Carrington JC, Vance VB (1997) Plant viral synergism: the potyviral genome encodes a broad-range pathogenicity enhancer that transactivates replication of heterologous viruses. Plant Cell 9: 859-868

Pruss GJ, Lawrence CB, Bass T, Li QQ, Bowman LH, Vance V (2004) The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens. Virology 320: 107-120

Punta M, Coggill PC, Eberhardt RY, Mistry J, Tate J, Boursnell C, Pang N, Forslund K, Ceric G, Clements J, Heger A, Holm L, Sonnhammer ELL, Eddy SR, Bateman A, Finn RD (2012) The Pfam protein families database. Nucleic Acids Res 40: D290-D301

Qi D, Omarov RT, Scholthof K-BG (2008) The complex subcellular distribution of satellite panicum mosaic virus capsid protein reflects its multifunctional role during infection. Virology 376: 154-164

Qiu W, Scholthof K-BG (2004) Satellite panicum mosaic virus capsid protein elicits symptoms on a nonhost plant and interferes with a suppressor of virus-induced gene silencing. Mol Plant Microbe Interact 17: 263-271

Qiu WP, Scholthof K-BG (2001) Genetic identification of multiple biological roles associated with the capsid protein of satellite panicum mosaic virus. Mol Plant Microbe Interact 14: 21-30

Rakhshandehroo F, Takeshita M, Squires J, Palukaitis P (2009) The influence of RNA-dependent RNA polymerase 1 on Potato virus Y infection and on other antiviral response genes. Mol Plant Microbe Interact 22: 1312-1318

Reddy ASN (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annu Rev Plant Biol 58: 267-294

Robaglia C, Caranta C (2006) Translation initiation factors: a weak link in plant RNA virus infection. Trends Plant Sci 11: 40-45

Rochow WF, Ross AF (1955) Virus multiplication in plants doubly infected by potato viruses X and Y. Virology 1: 10-27



Mandadi, p. 50

Rochow WF, Ross AF, Siegel BM (1955) Comparison of local-lesion and electron-microscope particle-count methods for assay of potato virus X from plants doubly infected by potato viruses X and Y. Virology 1: 28-39

Rushton PJ, Somssich IE, Ringler P, Shen QJ (2010) WRKY transcription factors. Trends Plant Sci 15: 247-258

Saeed A, Sharov V, White J, Li J, Liang W, Bhagabati N, J B, Klapa M, Currier T, Thiagarajan M, Sturn A, Snuffin M, Rezantsev A, Popov D, Ryltsov A, Kostukovich E, Borisovsky I, Liu Z, Vinsavich A, Trush V, Quackenbush J (2003) TM4: a free, open-source system for microarray data management and analysis. Biotechniques 34: 374-378

Satoh K, Kondoh H, Sasaya T, Shimizu T, Choi I-R, Omura T, Kikuchi S (2010) Selective modification of rice (Oryza sativa) gene expression by Rice stripe virus infection. J Gen Virol 91: 294-305

Scholthof K-BG (1999) A synergism induced by satellite panicum mosaic virus. Mol Plant Microbe Interact 12: 163-166

Scholthof K-BG, Jones RW, Jackson AO (1999) Biology and structure of plant satellite viruses activated by icosahedral helper viruses. Curr Top Microbiol Immunol 239: 123-143

Shimizu T, Satoh K, Kikuchi S, Omura T (2007) The repression of cell wall- and plastid-related genes and the induction of defense-related genes in rice plants infected with Rice dwarf virus. Mol Plant Microbe Interact 20: 247-254

Shiu S-H, Karlowski WM, Pan R, Tzeng Y-H, Mayer KFX, Li W-H (2004) Comparative analysis of the receptor-like kinase family in Arabidopsis and rice. Plant Cell 16: 1220-1234

Shpaer EG, Robinson M, Yee D, Candlin JD, Mines R, Hunkapiller T (1996) Sensitivity and selectivity in protein similarity searches: A comparison of Smith-Waterman in hardware to BLAST and FASTA. Genomics 38: 179-191

Sill WH, Pickett RC (1957) A new virus disease of switchgrass, Panicum virgatum L. Plant Dis Reptr 41: 241-249

Smith TF, Waterman MS (1981) Identification of common molecular subsequences. J Mol Biol 147: 195-197

Syller J (2012) Facilitative and antagonistic interactions between plant viruses in mixed infections. Mol Plant Pathol 13: 204-216

Tada Y, Spoel SH, Pajerowska-Mukhtar K, Mou Z, Song J, Wang C, Zuo J, Dong X (2008) Plant immunity requires conformational charges of NPR1 via S-Nitrosylation and thioredoxins. Science 321: 952-956

Thomas PD, Kejariwal A, Campbell MJ, Mi H, Diemer K, Guo N, Ladunga I, Ulitsky-Lazareva B, Muruganujan A, Rabkin S, Vandergriff JA, Doremieux O (2003) PANTHER: a browsable database of gene products organized by biological function, using curated protein family and subfamily classification. Nucleic Acids Res 31: 334-341

Turina M, Desvoyes B, Scholthof K-BG (2000) A gene cluster encoded by Panicum mosaic virus is associated with virus movement. Virology 266: 120-128

Turina M, Maruoka M, Monis J, Jackson AO, Scholthof K-BG (1998) Nucleotide sequence and infectivity of a full-length cDNA clone of Panicum mosaic virus. Virology 241: 141-155



Mandadi, p. 51

van der Fits L, Memelink J (2001) The jasmonate-inducible AP2/ERF-domain transcription factor ORCA3 activates gene expression via interaction with a jasmonate-responsive promoter element. Plant J 25: 43-53

van der Graaff E, Schwacke R, Schneider A, Desimone M, Flügge U-I, Kunze R (2006) Transcription analysis of Arabidopsis membrane transporters and hormone pathways during developmental and induced leaf senescence. Plant Physiol 141: 776-792

Van Valen L (1973) A new evolutionary law. Evol Theory 1: 1-30 Vance VB (1991) Replication of Potato virus X RNA is altered in coinfections with Potato virus

Y. Virology 182: 486-494 Verhage A, van Wees SCM, Pieterse CMJ (2010) Plant immunity: It’s the hormones talking,

but what do they say? Plant Physiol 154: 536-540 Vlot AC, Dempsey DMA, Klessig DF (2009) Salicylic acid, a multifaceted hormone to combat

disease. Annu Rev Phytopath 47: 177-206 Vogel J, Hill T (2008) High-efficiency Agrobacterium-mediated transformation of

Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep. 27: 471-478 Vogel JP, Garvin DF, Leong OM, Hayden DM (2006) Agrobacterium-mediated

transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss. Organ Cult. 84: 199-211

Wang D, Amornsiripanitch N, Dong X (2006) A genomic approach to identify regulatory nodes in the transcriptional network of systemic acquired resistance in plants. PLoS Pathog 2: e123

Wasternack C (2007) Jasmonates: An update on biosynthesis, signal transduction and action in plant stress response, growth and development. Ann Bot 100: 681-697

Whitham SA, Quan S, Chang H-S, Cooper B, Estes B, Zhu T, Wang X, Hou Y-M (2003) Diverse RNA viruses elicit the expression of common sets of genes in susceptible Arabidopsis thaliana plants. Plant J 33: 271-283

Whitham SA, Yang C, Goodin MM (2006) Global impact: elucidating plant responses to viral infection. Mol Plant Microbe Interact 19: 1207-1215

Xie Z, Fan B, Chen C, Chen Z (2001) An important role of an inducible RNA-dependent RNA polymerase in plant antiviral defense. Proc Natl Acad Sci USA 98: 6516-6521

Yu D, Chen C, Chen Z (2001) Evidence for an important role of WRKY DNA binding proteins in the regulation of NPR1 gene expression. Plant Cell 13: 1527-1540

Zhang X-C, Gassmann W (2003) RPS4-mediated disease resistance requires the combined presence of RPS4 transcripts with full-length and truncated open reading frames. Plant Cell 15: 2333-2342

Zhou N, Tootle TL, Tsui F, Klessig DF, Glazebrook J (1998) PAD4 functions upstream from salicylic acid to control defense responses in Arabidopsis. Plant Cell 10: 1021-1030



Stage I Stage II Stage III

DPI ≤ 7 d 10-14 d ≥21 d

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Figure 1. SPMV exacerbates disease symptoms and enhances PMV accumulation in Brachypodiumdistachyon. (A, B). Typical disease symptoms and gross effects of PMV alone and PMV+SPMV co-infection at 42 days-post-inoculation (dpi). PMV alone infection causes mild chlorosis, necrosis and stunting of roots and shoots, while PMV+SPMV co-infection causes severe stunting, chlorosis, and reduced seed-set. The inset image in “A” shows the severe chlorosis associated with co-infection with SPMV. The progression of PMV and PMV+SPMV infection can be classified into: (C) stage I with no visible chlorosis on leaves, stage II with mild chlorosis on leaves, and stage III with prominent chlorosis and necrosis on leaves, stunting and reduced seed-set. Immunoblot analysis of capsid protein accumulation at infection stages I, II and III with (D) PMV alone or (E) PMV+SPMV infection. The earlier detection of PMV CP in the mixed infections (E) is associated with SPMV enhancement of either PMV replication or movement, which is a component of the working definition of the SPMV-associated synergism (Scholthof, 1999). The asterisks (*) represent lower molecular-weight CPs, possibly produced from leaky scanning (Omarov et al., 2005) or post-translational modifications. The PonceauS stained nitrocellulose membranes serve to indicate approximately equal protein loading among the sample lanes.



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Figure 2. Brachypodium distachyon infections with PMV+SPMV compared to mock-inoculated plants.Typicaloutcomes of PMV+SPMV infection result in (A) leaf chlorosis and necrosis (indicated by arrows), (B) stunting, (C) reduced chlorophyll A and chlorophyll B (Chl A and Chl B) accumulation by stage III, (D) decreased fresh weight (FW) and dry weight (DW), (E) shorter internodes, (F) smaller leaves, but no significant effect on tiller number (G). Asterisks in the graph represent significant differences compared to mock controls (p-value <0.01, student t-test). (H) Immunoblot assays of PMV CP and SPMV CP accumulation in PMV+SPMV infected roots and shoots at stage III. The asterisk (*) represents a lower molecular-weight SPMV CP (see legend of Fig 1 and (Omarov et al., 2005). The Coomassie Brilliant Blue R-stained gel reflects approximate equal sample loading among the mock- and virus-inoculated sample lanes.



PMV PMV+SPMV

Up-regulated

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B

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Up-regulated Down-regulated

Figure 3. PMV and SPMV alter genes in diverse functional categories. Triplicate samples of Brachypodium (Bd21-3) plants infected with PMV or PMV+SPMV, or mock-inoculated at stage II (mild chlorosis symptoms) were analyzed using microarrays. Significant changes in gene expression relative to mock were determined using the Benjamini and Hochberg (BH) method by correcting for false discovery rate (q-value <0.05). (A) The Venn diagrams represent the number of genes up-regulated and down-regulated in response to PMV and PMV+SPMV infection. (B) Summary of the biological processes overrepresented in PMV- and PMV+SPMV-infections as deduced using AgriGO (Du et al., 2010), Pfamand PANTHER functional annotations (Thomas et al., 2003; Punta et al., 2012). A detailed list of the GO assignments and SEA analysis is in Table S2.



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Log2B

Figure 4. Pseudo-colored heat map representation of Brachypodium gene expression in PMV alone and PMV+SPMV infection. The color bars on the top indicate logarithmic expression values (log2) from the microarray; green for lower expression and red for higher expression. (A) The up-regulated glutathione S-transferase proteins, chaperone/heat shock proteins (HSPs), cytochrome P450 family proteins, and several genes implicated in protein synthesis are indicated. (B) The down-regulated photosynthesis-related genes are indicated (see also Figure 2C for effects on chlorophyll content). Functional classification of the different genes was deduced using AgriGO (Du et al., 2010), Pfam, and PANTHER annotations (Thomas et al., 2003; Punta et al., 2012). Note that genes with multiple alternatively spliced transcripts occur more than once, and certain alternatively spliced transcripts exhibit differential expression kinetics.



B

A

Group IIa

Group IC

Group III

Group IIc

Bradi4g30360 150 SPLRDDGTYRRIKVKRVCTRIDPSDTSLVVKDGYQWRKYGQKVTRDNPSPRAYFRCAF--

Bradi2g30695 85 SS---RGACR----RRTQQSSVVTKTMNTLDDGQAWRKYGQKYIHNSKHPRAYFRCTHKYBradi2g15877 3 RK---RGAME---KVRRQVRVSSVHDLAPLDDGLSWRKYGQKDILGAKYPRAYFRCTHRN

Bradi4g19060 93 KK---KGEKK---ERRPRYAFQTRSQVDILDDGYRWRKYGQKAVKNNNFPRSYYRCTH--Bradi2g05510 126 -E---LDAAA---RGHRRIGFRTRSAVEVMEDGFRWRKYGKKAVKSSPNLRNYYRCSA--consensus ... . .. . . ...**. *****.* .. .*.*.**..

Bradi4g30360 208 APSCPIKKKVQRSA---ENSSVLEATYEGEHNHPQPTRAGELTSSCVTRSGSVPCSISINBradi2g30695 138 DQQCAAQRQVQRCEDDDDDKDTFRVTYIGVHTCRDPAAAPVHVTRTA------GCHLISF

Bradi2g15877 57 TQGCQATKQVQRD---AGDPLIFDVVYHGDHTCAQGLQHLQRHDQRQ-------------Bradi4g19060 145 -QGCNVKKQVQRLS---RDEGVVVTTYEGTHTHPIEKSNDNFEHILT------Q------

Bradi2g05510 177 -PGCGVKKRVERDR---HDPAYVITTYHGVHNHPTPAAS*--------------------consensus ..* ....*.* . .. .* * *... .

Figure 5. Sequence analysis of PMV and SPMV modulated WRKY transcription factors in Brachypodium. (A) Multiple sequence alignment of WRKY-domain sequence of WRKY factors mis-expressed in PMV and SPMV infection. The consensus symbols are indicated below the alignments with identically conserved residues indicated by black shading and an asterisk (*). Amino acids with ≥ 50% identity are shaded grey and marked with a period (•). The conserved WRKY and zinc-finger motifs are underlined and in red and blue, respectively. (B) The phylogenetic relationship of Brachypodium WRKY factors mis-expressed in PMV and PMV+SPMV infections with orthologous Arabidopsis WRKY factors. The protein sequences were retrieved from Phytozome(Goodstein et al., 2012), aligned using ClustalX2 (Larkin et al., 2007), and displayed as an unrooted tree using Archeopteryx (Han and Zmasek, 2009). The branch length at bottom right of the phylogram indicates genetic distances. Down-regulated genes are identified by black triangles. The grouping of WRKY factors into Group Ic, IIa, IIc and III was based on the classification described previously (Rushton et al., 2010).



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Figure 6. Temporal expression analysis of SA, JA and ET components during PMV and SPMV disease progression. Brachypodium plants infected with PMV, PMV+SPMV, or mock-inoculated sampled at stages I, II and III (see Figure 1C for description of stages) to determine the expression of putative genes in SA responses (A) PR-1-like, (B) PR-3-like, (C) PR-5-like, (D)PAD4-like and (E) AOX1A; JA responses (F) LOX2, (G) AOS and (H) OPR3; and, ET responses (I) ERF-1and (J) ERF3-like using (q) RT-PCR analysis. Transcript level of UBIQUITIN18(UBC18) was used to normalize the (q) RT-PCR data. Expression values plotted are average of three biological replicates. Error bars represent standard deviation among the replicates. Significant changes in gene expression was determined using Student’s t-test (p-value <0.05). Note that the expression scales for each gene panel are unique and are relative to the mock-inoculated control plants at stage I. Primers used are listed in Table S7



Figure 7. Phylogenetic classification of Brachypodium serine-threonine kinases altered in PMV and PMV+SPMV infection. The sequences were retrieved from Phytozome (Goodstein et al., 2012), aligned using ClustalX2 (Larkin et al., 2007), and displayed as an unrooted tree using Archeopteryx (Han and Zmasek, 2009). The functional domains and sub-families of the kinases were deduced using predicted Pfam and PANTHER annotations (Thomas et al., 2003; Punta et al., 2012) using Phytozome (Goodstein et al., 2012). The kinases were grouped into subfamilies: LRR-VIII-2, LRR-XII , WAK/LRK10L-1, RLCK-OS2, RLCK-IV, RLCK-IXb, SD-1c , SD-2b, MEKK_ste11_MAP3K , KIN1/SNF1/Nim1_like (CAMK_2), PVPK_like_kin82y (AGC_8), L-LEC, and DUF26, following a classification previously described (Shiu et al., 2004). Down-regulated genes are identified by black triangles. Note that among the down-regulated genes, Bradi2g47510, Bradi4g30380, and Bradi4g45310 cluster together, suggesting a transcriptional co-regulation of these phylogenetically-related kinases.



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Figure 8. Proposed model for the host:pathogen interactions induced by PMV alone or the PMV+SPMV synergism in Brachypodium. Briefly, the PMV replicase-associated proteins (P48, P112), movement-associated proteins (P8, P6.6, P15 ), PMV capsid protein (CP) and SPMV capsid protein (SPCP) cooperatively function in translation, replication, encapsidation, and movement of PMV and SPMV in a systemic infection of Brachypodium and other grasses in the Poaceae. PMV and SPMV move cell-to-cell primarily via plasmodesmata (up-down arrow) as viral ribonucleoprotein(vRNP) complexes, probably with assistance from PMV P8, SPCP (which are also associated with the host cell-membrane), P6.6, and P15 (Turina et al., 2000; Qi et al., 2008). Blue and orange beads on a string represent PMV and SPMV vRNPs, respectively. Both PMV virions (blue hexagon) and SPMV virions (orange hexagon) assemble in the cytosol of an infected leaf and are probably introduced into new cells through cell damage (lightning bolt). An as of yet unidentified PMV- or SPMV-associated viral component (RNA and/or proteins indicated by “?”) elicits host defense responses activating expression of multiple transmembrane receptor-like kinases (TM-RLK) and cytoplasmic receptor-like kinases (Cyto-RLK). PMV and PMV+SPMV infection also triggers expression of salicylic acid (SA)-, jasmonic acid (JA)-, and ethylene components, possibly as a host defense response (the red and green text indicates up- and down-regulated genes, respectively). SPCP also localizes to the host nucleus and nucleolus (Qi et al., 2008) thereby uniquely modulating expression of putative splicing factors (SF1 and SF3A) and an AP2 factor and may affect alternative splicing of certain host mRNAs. The annotation of Brachypodium genes is based on orthologous Arabidopsis proteins. Further outcomes of the Brachypodium transcriptome changes in response to PMV and PMV+SPMV infections are explained in the Discussion. The PMV and SPMV genomes are shown in Figure S1.



running head: antiviral responses in the model monocot ... · 06.09.2012 · pmv include foxtail...

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