developing a sustainable management strategy to …

DEVELOPING A SUSTAINABLE MANAGEMENT STRATEGY TO

CONTROL WALNUT BLIGHT

Cintia Sagawa, Renata Assis, Shijao Jiang, Bipin Balan, Aaron Jacobson, Sriema Walawage Paulo

Zaini and Abhaya Dandekar.

ABSTRACT

Walnut blight is a bacterial disease that reduces the productivity of walnut orchards and negatively

influences the quality of the nuts, especially in wet years and on early leafing varieties. Walnut

blight is caused by Xanthomonas arbicola pv. juglandis, (Xaj) which attacks various tissues of

English walnut including catkins, female flowers, fruit, green shoots, buds, and leaves. Significant

economic loss occurs mainly from infection of fruit soon after flowering. The extended period of

host susceptibility is one of the chief obstacles to controlling the disease. However, we do not

know much of the biology of the host-pathogen interactions and more specifically about

underlying factors that mediate virulence or increased susceptibility and/or resistance of the host

plant. Current measures involve the use of copper-based biocides and ethylene bis-dithiocarbamate

(EBDC) fungicides to control walnut blight disease. This investigation focuses on developing

novel approaches based on genomic information to control the disease focusing on two aspects. 1)

Evaluating the genome content of the pathogen Xanthomonas arbicola to deepen our

understanding of how the pathogen interacts with the plant apoplast to cause the disease, and 2)

dissecting the host-pathogen cellular responses to define how the secreted pathogen proteins

promote the entry and propagation of the pathogen leading to disease development. We are

dissecting how these proteins are early biomarkers of disease development and trigger host

determinants that regulate the host susceptibility to walnut blight disease.

OBJECTIVES

Objective 1: Evaluate the genome content of the pathogen to discover pathogenic attributes related

to the development of walnut blight.

Objective 2: Dissecting the host-pathogen response to define early biomarkers of disease to define

host determinants that regulate the host susceptibility to walnut blight disease.

SIGNIFICANT FINDINGS



• Xaj prefers to enter through stomatal openings and to colonize the apoplast of mesophyll

cells

• Xaj secretes a battery of proteins that can degrade plant cell walls

• Xaj is able to metabolize phenols and suppress plant immunity

• Xaj is able to metabolize methanol and potentially trigger oxidative stress



• Xaj mutated for 3 of the 7 secreted protein are less pathogenic indicating that the secretory

proteins play a significant early role in blight disease development.

• We have identified 25 SWEET proteins that are good early biomarkers for regulating

susceptibility to walnut bacterial blight.

PROCEDURES



The walnut blight pathogen, Xanthomonas arboricola pv. juglandis strain 417 used in our study

was sequenced and the whole genome sequence information was deposited in GenBank (NCBI

accession number CP012251; Pereira et al., 2015). This genome sequence is 5,218,943 nucleotides

in length and encodes 4,178 putative genes was also uploaded to the antiSMASH website so as to

be accessible for custom analysis that are available online at

https://antismash.secondarymetabolites.org/#!/start to identify novel and common gene clusters

found exclusively in Xaj-417. To identify common gene clusters in Xaj-417 we compared its

sequence with the genomes of other related bacterial to identify specific genes related to this

disease. The Blast algorithm within the Orthologsorter program

(http://jau.facom.ufms.br/arboricola/; Farias and Almeida, 2013) was used to make the

comparisons. The first comparison included all the plant pathogen genomes within the

Xanthomonadaceae family present in the NCBI database, which included 55 strains of the genus

Xanthomonas and 7 strains of the genus Xylella. We were successful in the identification of 7

highly conserved secreted proteins (Fig 1; Assis et al 2017). A comparison of the Xaj-417 genome

with 226 proteobacterial genomes revealed the presence of 64 TonB receptor (TNBR) type loci in

Xaj-417 necessary for the utilization of carbohydrates and phenols (Fig 1). Orthologsorter program

was also used to identify unique genes exclusively present in Xaj-417 by making comparisons to

20 more closely related X.arboricola strains (Fig 2) and then to 5 Xaj strains (Fig 3).

We were successful in linking metabolic traits to phenolic pathway genes by measuring the growth

response of Xaj-417 to specific pathway intermediates. The phenolic compounds in a particular

pathway served as a sole carbon source to support the growth of the bacteria. The bacteria were

grown overnight in minimal media supplemented with glucose (20%) as a sole carbon source, the

tubes were centrifuged, the pellet was washed and the optical density (OD) adjusted to 108 cells/ml.

About 200ul of culture was used as inoculum for 5 mL M63 + 1mM phenolic compound and

grown for 15 days. Growth was monitored by measuring OD periodically and the viability of the

cells was evaluated using the L/D BacLight kit where SYTO9 and PI stains was added to 150 ul

of culture. The fluorescence emitted was measured using a microplate reader to measure the

percentage of live versus dead bacterial cells (Fig 4A). We used various carbon sources to test the

functionality of various pathway genes and included phenolic compounds (Fig 4B), sugars that are

breakdown products of cell wall degradation and methanol. The bacteria were fixed and visualized

by microscopy to evaluate the morphology of the growing bacterial cells.

https://antismash.secondarymetabolites.org/#!/start

http://jau.facom.ufms.br/arboricola/



To define host determinants for susceptibility to walnut blight we followed two independent

investigative paths, 1) Created mutations in some of the key genes identified by the bioinformatics

analysis that we conducted of the pathogen genome and 2) identified key susceptibility genes by

doing a bioinformatics analysis of the Juglans regia version 1.0 genome sequence. Mutations were

created by insertion-based knock-out in three of key virulence genes identified by bioinformatics

analysis presented in objective 1. Two of the 7 genes, CM, and argG were targeted for gene

disruption this year. The procedure to isolate mutants in these genes was described in last year’s

report for lesA (Nascimento et al., 2016). Xaj-417’s lesA gene sequence was synthesized using

chemical synthesis and optimized for expression in E.coli as well as was interrupted at one of the

active site residues with a functional gene encoding kanamycin resistance as previously described

(Nascimento et al 2016). CM and argG mutant strains were also obtained by synthetic gene

synthesis with a knanamycin resistance gene inserted into the active residue site of the protein

resulting in a non-functional enzyme. The plasmid containing the new version was electroporated

into Xaj 417 cells for replacement of the gene by homologous recombination (Matsumoto et al

2012). Kanamycin resistant colonies were tested for gene disruption after growth on media

containing kanamycin (Fig 5; Nascimento et al., 2016). Mutant lines were confirmed by growth

on media containing kanamycin and by analysis of genomic DNA to confirm the presence of the

inserted DNA in the gene, CM or argG (Fig 5).

To test for bacterial virulence, we used plants overexpressing PPO genes as sensitive disease

indicators as described last year. Micro-propagated shoots of high expressing JrPPO1gene (8H and

11E lines), were inoculated with 30 mL of D/W solution with 106 cells/mL of Xaj-417 mutants for

10 minutes. The formation of black spots was monitored during 5 days post inoculation (d.p.i.)

(Figure 7). After 10 d.p.i, shoots were weighted and incubated in 20 mL of D/W containing 0.01%

of Tween 20 with agitation for 40 min for release of bacteria from infected tissues. Bacterial titers

were estimated in the solution by plating out serial dilutions as earlier described (Lindow et al.,

2014, McGuire and Jones, 1986). Another test was performed by comparing difference in the

disease symptoms caused by the inoculation of each bacterial mutant and wild type on healthy nuts

harvested from the field in Aug 2018. The nuts were immersed in a bacterial solution of 107

cells/ml in sterile water with MgCl2 and surfactant (Breakthrough) for 20 min. The nuts were then

incubated in a sweater box at 100% humidity for 1 week. Nuts examined for blight symptoms (Fig

7) and for the location of the bacteria using a scanning electron microscope using pieces of hull

tissues (Fig 6).

We used two different approaches to identify walnut SWEET genes using version 1 of the whole

Juglas regia genome annotation available online from the National Centre for Biotechnology

Information (NCBI; https://www.ncbi.nlm.nih.gov). In the first approach, we obtained putative

SWEET genes by searching hidden Markov Models (HMMER. Available online:

http://hmmer.org) profiling the core domain of MtN3/salivary protein (PF03083) from Pfam

database (Pfam. Available online: http://pfam.xfam.org), 37 putative proteins were matched with

an E-value cutoff of 0.0001. In the second approach, we retrieved all 17 known SWEET proteins

of A. thaliana (Chen et al, 2010) from Arabidopsis Information Resource (TAIR. Available online:

https://www.arabidopsis.org/), putative walnut SWEET proteins were identified by blast searches

https://www.ncbi.nlm.nih.gov)/

http://hmmer.org)/

http://pfam.xfam.org)/

https://www.arabidopsis.org/)

against walnut genome using A. thaliana SWEET proteins sequences as queries. We were able to

retrieve 28 candidate walnut SWEET proteins. To confirm the completeness of the core domain

(s) of these candidate proteins we manually verified with the Conserved Domain Database

(CDD, Available online: https://www.ncbi.nlm.nih.gov/cdd ) and SMART (SMART. Available

online:http://smart.embl-heidelberg.de/). Some candidates were abandoned, as they were too short

and/or incomplete. Finally, 25 non-redundant proteins and their gene sequence were selected and

named based on their position on pseudomolecules of the Walnut Genome version 1.

RESULTS



The walnut blight pathogen, Xanthomonas arboricola pv. juglandis strain 417 (Xaj-417) genome

was compared with 53 genomes of Xanthomonas and 7 genomes of the genus Xylella all known

and well described plant pathogens. Common to all of these plant pathogens are 7 secreted proteins

that are potential virulence factors (Fig 1). The unique genes for each comparisons were also

revealed, 136 for Xaj-417, 171 for Xanthomonas and 136 for Xylella. A comparison with 226

proteobacterial genomes revealed the presence of 72 TonB receptor (TNBR) type loci in

Xanthomonas, 8 of these are common with Xylella pathogens. The number of unique genes in Xaj-

417 drops to 54 when comparing all 20 genomes of X.arboricola and increases to 140 when

comparing to the 4 other juglandis genomes in the NCBI database. Most notable among the unique

genes present in Xaj-417 are genes for nitrate metabolism, resistance to copper, genes involved in

non-ribosomal protein synthesis (NRPS), a type 4-secretion system (T4SS). We have begun

performing growth and viability assay to validate the TNBR loci with the utilization of

carbohydrates and phenols. Two phenol degradative pathways were investigated vanillic acid

(VA) and 4-hydroxy benzoic acid (HBA). Shown in Fig 4 is growth of Xaj-417 on HBA but cannot

utilize benzoic acid (BA). Growth on HBA is much slower as compared to when glucose is used

as the sole carbohydrate (Fig 4). We are currently investigating other carbohydrates that are

breakdown products of the plant cell walls with special emphasis on pectin products. The

distribution Xaj-417 was examined by scanning EM of infected tissues and the bacteria seem to

colonize the stomata openings and were on the inside the leaf (Fig 6). Methanol is one of the

products found in leaves and Xaj-417 could grow on it as a sole carbon source. We were also able

to isolate a pink bacterial isolate (PBI) from walnut vegetative buds obtained from the field. We

have 4 PBIs (PBI-1, PBI-2, PBI-3 and PBI-4) and they all can grow on methanol as a sole carbon

source. We are currently performing a DNA sequence analysis of these bacteria to determine the

species and type of bacteria. We would like to understand its association with Xaj-417 and

potential role on Walnut Blight development.



In our last year report, we had reported the successful knock out of the gene encoding LesA from

Xaj-417 one of the common secreted virulence factor encoded in this organism. Since then, we

have created two additional knock out lines of Xaj-417, one for the chorismate mutase (CM)

encoding gene and the other that encodes argininosuccinate synthase (ArgG). Mutant lines were

https://www.ncbi.nlm.nih.gov/cdd%20)

http://smart.embl-heidelberg.de/)

confirmed by growth on media containing kanamycin and by analysis of genomic DNA to confirm

the presence of the inserted DNA in the gene, cm or argG (Fig 5). To further define the role of

these common virulence factors in walnut blight the mutant bacteria were used to infect nuts

obtained from the field and the PPO1 shoot cultures. In our last year’s report, we showed how the

PPO1 over expressing shoot cultures were sensitive to infection by Xaj. The Xaj-lesA-, Xaj-cm-

and Xaj-argG- were compared to mock infected and Xaj-417 (Xaj-WT) nuts or shoots. In all cases

the mock infected showed no disease symptoms while Xaj-WT infected tissues showed significant

disease symptoms. The disease incidence was much less for Xaj-lesA- or Xaj-cm- as compared to

Xaj-WT infections (Fig 7). The disease incidence for Xaj-argG- was significantly less to that

observed for the Xaj-lesA- or Xaj-cm- strains (Fig. 7). We have been successful in carrying out a

bioinformatics analysis to identify walnut host SWEET genes that encode a pathogen triggered

sucrose translocator. We have been able to identify 25 SWEET genes and a phylogenetic analysis

reveals strong similarity to previously identified SWEET genes from rice and Arabidopsis that

have been shown to be involved in increasing sensitivity to pathogens (Fig 8A). We were able to

find expression data for 21 of the 25 SWEET genes and the expression pattern of these 21 genes

is shown in a heat map that includes RNAseq data of 20 different walnut tissues (Fig 8B).

DISCUSSION

To deepen the understanding of the pathogen-host factors essential to the causation of walnut

blight disease we have analyzed both the pathogen and host genomes to identify virulence factors

and host susceptibility genes. To define pathogen factors we took an unbiased approach to our

analysis and we analyzed all 4,178 putative genes in the Xaj-417 genome to sift out genes

responsible for pathogenesis leading to disease. Xaj attacks various tissues of English walnut

including catkins, female flowers, fruit, green shoots, buds and leaves; however, we do not know

how this occurs. Our SEM analysis revealed that Xaj preferentially colonizes stomata and in leaves

and fruit where it is able enter on the inside of these tissues through the stomatal openings (Fig 6).

Our comparison to all known plant pathogens within Xanthomonadeacea revealed seven common

proteins. These seven proteins are secreted into the plant apoplast via Xaj-417’s type 2 secretion

system (T2SS). Most plant pathogens use the T2SS to secrete hydrolytic enzymes to aid in the

colonization phase. To further define the role of these proteins in the disease process we created

mutants of Xaj-417 that were unable to express one of these proteins. LesA is a secreted lipase

was the first candidate to be analyzed as it has been shown to be essential for virulence in

grapevines infected by Xylella (Nascimento et al., 2016; Gouran et al., 2016) or in rice infected

with Xoo (Aparna et al., 2009). In all of these studies, it has been shown that knockouts of LesA

are less or non pathogenic. This also appears to be the case with the lesA knock out of Xaj-417

that is much less virulent as compared to the wild type Xaj-417. The second secreted protein

investigated was a secreted chorismate mutase CM that converts chorismate, a common

intermediate in the plant phenolic pathway essential for the synthesis of salicylic acid and auxin

both necessary for the plant to mount an immune defense against pathogen attack. Our analysis

indicates that Xaj-417 uses CM to cause an immune deficiency in the plant and it converts

chorismate into a siderophore that it uses to sequester iron (Fe). A mutant deficient in the

production of CM, Xaj-CM- is much less virulent as compared to Xaj-WT (Fig 7), indicating that

the secreted form of CM is a virulence factor made by the bacteria to cause disease in walnut.

Arginosuccinate synthase (argG) is a key virulence factor in both Xylella and Xanthomonas

required for the synthesis of the amino acid arginine. The argG in Xaj-417 was knocked-out by

insertional mutagenesis (Fig. 5). Xaj-argG- mutants showed much lower incidence of disease,

much lower than observed for LesA or CM mutants. This indicates that ArgG plays an important

role in the symptoms associated with walnut blight disease.

To define host plant determinants that encode for susceptibility to walnut blight we focused on

sucrose efflux translocators (SWEET) proteins. The efflux of sucrose to the apoplast is key as it

provides an excellent carbon source to support the growth of Xaj in the apoplast. Sucrose is a

product of photosynthesis in the mesophyll cells and is typically translocated to phloem via a

similar set of SWEET proteins. In rice, it has been shown that Xoo encodes TALEN proteins that

are injected into the plant cell via a T3SS system and once inside the cell bind upstream to activate

a particular set of SWEET genes that translocate sucrose to the apoplast to feed the Xoo pathogen.

We identified by analysis of the Chandler version 1.0 genome that is encodes 25 SWEET proteins.

We conducted a phylogenetic analysis comparing these 25 proteins from Chandler with the 17

SWEET proteins from Arabidopsis and the 21 SWEET proteins from rice. Shown in Fig 8A is a

phylogenetic tree that shows the relationship between all 63 SWEET proteins, 25 are J.regia

Chandler, 21 rice and 17 Arabidopsis. These proteins fall into 4 clades, Clade 1 contains 8

Chandler SWEET proteins out of the 17, Clade to has 5 out of 19, Clade III has 6 out of 9 and

Clade 4 has 6 out of 18. Expression values of JreSWEET transcripts was extracted from RNA-seq

data of ‘Chandler’ that includes expression in twenty different tissues or developmental stages.

Transcript abundance was determined for thirteen anatomic tissues (callus, catkins, embryo,

pistillate-flower, hull, fruit, leaves, packing-tissue, pellicle, root, somatic embryo, wood and bud)

and developmental stages of five tissues (callus, hull, leaf, packing tissue and embryo) was used

for 21 of the 25 JreSWEET genes showed expression and were analyzed (Fig. 8B). The expression

data was used to generate heat map using Morpheus heat map program (MORPHEUS. Available

online: https://software.broadinstitute.org/morpheus/) package with hierarchical clustering

method for twenty different tissues (Fig. 8B). We need to confirm this data using real-time analysis

using RNA obtained from infected tissues and from proteomics experiments and these are planned

for this coming year.

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Figure 1: Identification of common and unique genes present in the genome of Xanthomonas

arboricola pv. juglandis strain 417 when compared to the genomes of Xanthomonadeaceae plant

pathogens.

Figure 2: Phylogenetic analysis of 20 Xanthomonas arboricola strains present in NCBI and their

relationship to Xanthomonas arboricola pv. juglandis strain 417.

Figure 3: Identification of common and unique genes present in the genome of Xanthomonas

arboricola pv. juglandis strain 417 when compared to the genomes of four other juglansis strains

present in NCBI.

Fig 4: Measurement of growth in the presence of phenolic substrates and measurement of live vs

dead bacteria. A) measurement of the proportion live bacteria by integrating the intensities of the

green (live; 510–540 nm) and red (dead; 620–650 nm) emission were acquired, and the green/red

fluorescence ratios were calculated for each proportion of live/dead Xaj. B) Growth curve of Xaj

in minimal media supplemented with phenolic compound.

Fig 5: Targeting the argG gene of Xaj-417 for mutagenesis. A) The arg loci in Xaj-417

highlighting the argG gene. B) The arg pathway and associated genes in Xanthomonas. C) Gel

confirming the insertion of the kan gene in the argG gene.

Fig 6: Micrographs of Xaj 417 WT in A) bacterial cells from agar cultures and in B) stomata

opening in a symptomatic walnut nut surface.

Fig 7: Walnut blight disease phenotyping in nuts and shoots. A) Walnut fruits were inoculated

with 107 bacterial cells/mL in a solution of MgCl2 and incubated in a humid box at room

temperature for 7 days. Walnut shoots inoculated with Xaj-417 WT and the less virulent strain

Xaj-417 argG- and incubated at RT for 7 days.

Fig 8: Unrooted neighbor-joining phylogenetic tree of SWEET proteins in Arabidopsis, rice and

walnut. Different color represented four different clades, which clade Ⅰ in blue, clade Ⅱ in red,

clade Ⅲ in green and clade Ⅳ in black. Scale bar represent 0.1 substitutions per amino acid

position.

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