LARGE-SCALE BIOLOGY ARTICLE

Fusarium virguliforme Transcriptional Plasticity Is Revealed byHost Colonization of Maize versus Soybean[OPEN]

Amy Baetsen-Youngab Ching Man Waibc Robert VanBurenbc and Brad Dayab1

a Department of Plant Soil and Microbial Sciences Michigan State University East Lansing Michigan 48824b Plant Resilience Institute Michigan State University East Lansing Michigan 48824cDepartment of Horticulture Michigan State University East Lansing Michigan 48824

ORCID IDs 0000-0001-6710-3192 (AB-Y) 0000-0002-2913-5721 (CMW) 0000-0003-2133-2760 (RV) 0000-0002-9880-4319(BD)

We exploited the broad host range of Fusarium virguliforme to identify differential fungal responses leading to either anendophytic or a pathogenic lifestyle during colonization of maize (Zea mays) and soybean (Glycine max) respectively Toprovide a foundation to survey the transcriptomic landscape we produced an improved de novo genome assembly andannotation of F virguliforme using PacBio sequencing Next we conducted a high-resolution time course of F virguliformecolonization and infection of both soybean a symptomatic host and maize an asymptomatic host Comparativetranscriptomic analyses uncovered a nearly complete network rewiring with less than 8 average gene coexpressionmodule overlap upon colonizing the different plant hosts Divergence of transcriptomes originating from host specifictemporal induction genes is central to infection and colonization including carbohydrate-active enzymes (CAZymes) andnecrosis inducing effectors Upregulation of Zn(II)-Cys6 transcription factors were uniquely induced in soybean at 2 dpostinoculation suggestive of enhanced pathogen virulence on soybean In total the data described herein suggest that Fvirguliforme modulates divergent infection profiles through transcriptional plasticity

INTRODUCTION

During host colonization fungal plant pathogens elicit an array ofsymptoms in the plant many of which stem not only from themodulation of the plant immune system but also from reprog-ramming of host development processes (Oliver and Ipcho 2004Horbach et al 2011 Cordovez et al 2017) Indeed while studiessurveying single trait interactions have highlighted key processesand pathways critical to pathogenesis of fungi in plants (Derntlet al 2017Fanget al 2017) genomic and transcriptomic studiessuggest fungi have a complex and elaborate infection program(Brown et al 2017 Chowdhury et al 2017) Comparative tran-scriptomics during fungal colonization have revealed that in-fection programs vary by fungal lifestyle suggesting that inducedpathways diverge within biotrophic hemibiotrophic andor ne-crotrophic interactions Moreover studies investigating the in-teractions of fungal isolates that elicit phenotypically distinct hostsymptoms have led to the discovery of numerous disparateprocesses bywhich fungi subvert host defenses (OrsquoConnell et al2012 Haueisen et al 2018) Indeed transcriptome-based ap-proaches have shown that fungi regulate both host and fungal

developmental programs to penetrate and colonize plants(Soaneset al 2012 Vollmeister et al 2012) use aconstellationofsecreted effector molecules (Yang et al 2013 Haueisen et al2018) and express small RNAs which modulate host defensesignaling (Jiang et al 2017 Lee Marzano et al 2018) Howevereach of these studies has focused on pinpointing candidates forvirulence or aggressiveness based on the interaction between thepathogen and a single host While obviously informative theoutcomes of these approaches have left significant gaps in ourunderstanding as tohow individual pathogens infect andor causedisease in multiple hostsFungal plant pathogens often have broad host ranges high-

lighting their ability to cause substantial economic losses in globalagricultural systems Although most plant pathogens colonizeonly a narrow range of host plants several fungi have broadpathogenic host ranges with additional endophytic host rangesthat involving symptomless host penetration and development(Derbyshire et al 2017) For example Verticillium dahliae causesdiseases on more than 400 different hosts and although it isadapted to specific hosts to cause disease it has a much largerasymptomatic endophytic host range (Malcolm et al 2013) Thisendophytic host range was more recently discovered as thecausal agent of soybean (Glycine max) sudden death syndrome(SDS) caused by Fusarium virguliforme (Kolander et al 2012)This shows that individual fungi can manipulate their geneticexpression programs to enable colonization of hosts with distinctpathogenic and endophytic outcomes Studying fungal specieswithbothbroadsymptomaticandasymptomatichostphenotypes

1Address correspondence to bdaymsueduThe author(s) responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (wwwplantcellorg) is Brad Day (bdaymsuedu)[OPEN]Articles can be viewed without a subscriptionwwwplantcellorgcgidoi101105tpc1900697

The Plant Cell Vol 32 336ndash351 February 2020 wwwplantcellorg atilde 2020 ASPB

provides an opportunity to understand the transcriptional re-programming required to promote fungal colonization of hostsand disease development

In silico comparative studies haveprovided sufficient resolutionto differentiate disease-eliciting plant pathogen interactions fromthose that are primarily endophytic (Laluk and Mengiste 2010Lofgren et al 2018) However fungal ecology-based analyses inBotrytis Verticillium and Fusarium species suggest that hostfungal interactions exhibit a continuum of molecular crosstalkThis results in a gradation of pathogenic to mutualistic outcomeswhen interacting with diverse hosts as demonstrated (Malcolmet al 2013 Demers et al 2015 Shaw et al 2016) Overall thesestudies demonstrate that at least in the case of the aforemen-tioned species fungi can fulfill two distinct ecological nichespotentially within the same community (Selosse et al 2018)Exploring the genomes of fungi with broad host ranges has un-covered the genomic potential that enables them to occupy di-verse ecological and pathogenic niches (Ma et al 2010 Seidlet al 2015 Derbyshire et al 2017)Weposit that a comparison ofthe underlying transcriptional processes regulating a pathogenicversus endophytic lifestyle will yield novel genetic signaturespromoting virulence within a susceptible host

F virguliforme the causal agent of soybean SDS is an ex-ceptionalmodel foranalyzing fungal-plant interactionsdueboth toits broad host range and to the severe economic loss it causes inthe soybean industry This disease is a key limitation in reachingsoybean yield potential with an estimated annual economic im-pact of $330 million in the United States partly stemming fromlimited effective disease management practices (Koenning andWrather 2010 Hartman et al 2015) F virguliforme is an asco-mycete that colonizes the roots of more than 10 plant speciesstimulating leaf chlorosis and root necrosis resulting in the

eventual loss of above ground biomass (Kolander et al 2012)However on many monocots and weed species F virguliformecolonizes roots with no observable deleterious phenotype in thehost (Kolander et al 2012 Kobayashi-Leonel et al 2017)F virguliforme is asymptomatic inmaize (Zeamays) and in thefieldmay form endophytic associations between crop rotations withsoybean in the same agroecosystem Given our lack of un-derstanding of how F virguliforme interacts with potential hostplants (eg soybean and maize) and subsequently occupiesdistinct ecological nichesweperformedasystematiccomparisonby investigating the host-pathogen transcriptomic interfaceduring symptomatic and asymptomatic colonization To highlightthe divergence in genetic signaling pathways underpinning fungallifestyle we investigated (1) the early stages of colonization be-tween F virguliforme and soybean and maize (2) how earlytranscriptional responses of F virguliforme colonizing maize orsoybean are regulated and (3) the potential conservation andordistinction between asymptomatic versus symptomatic fungaltranscriptomes

RESULTS

Generation of a High-Contiguity Reference Genome forF virguliforme

Although a draft genome of F virguliforme is available (Srivastavaet al 2014) the current version is incomplete thus limitingcomparative and functional analyses Therefore we generated animproved pathogen reference genome using third-generationPacBio single-molecule sequencing technologies We gener-ated a high-quality F virguliforme genome using173 coverage

Fungal Transcriptional Plasticity Between Hosts 337

of PacBio data (Supplemental Figure 1) Filtered reads were as-sembled using the long-read optimized assembler Canu (Korenet al 2017) and resultant contigswere error-corrected using 503Illumina data using Pilon (Walker et al 2014) Our assembledFvirguliformegenomeencompassed52MBwith96contigswithanN50 of 154 MB (Supplemental Table 1) The resultant genome size(Mb) was slightly larger than the version 1 (v1) draft assembly(Srivastava et al 2014) and in this study the contiguity and N50were significantly improved (Supplemental Table 1) Syntenybetween the two genome versions was highly fragmented(Figure 1A) perhaps a result of having more than 3000 contigs inthe first version of the genome Notably within syntenic regionsmicro-collinearity between the two genomes was highly con-served (Figure 1B)

The F virguliforme genome generated in this study was an-notated using FunGAP (Min et al 2017) incorporating AUGUS-TUS MAKER and BRAKER gene model prediction algorithms

(Stanke et al 2006 Cantarel et al 2008 Hoff et al 2016) A totalof 16050 genes from the F virguliforme version 20 (v2) genomewere discovered representing an increase of 1205 genes com-pared with version v1 Comparisons of the coding sequencesbetween two genome versions revealed 12306 conserved geneswith aminimum 70gene alignment rate and 95 identity 1422genes were considered as misassembled or incomplete in one ofthe genomes Overall 2889 new genes that were missing in v1were annotated in v2 and were considered novel (SupplementalData Set 1) This gene set was enriched with genes involved inprotein ubiquitination organic compound breakdown and por-phyrin compound biosynthesis (Supplemental Table 2) Nextthe completeness of genome annotation was evaluated usingBenchmarking Universal Single-Copy Orthologs (Simatildeo et al2015 Waterhouse et al 2018) and we observed an 98completionwith10162of the16050genesbeing supportedwithprotein evidenceFurther explorationof thegenomeusingSignalP(v41 Petersen et al 2011) and EffectorP (v20 Sperschneideret al 2016) discovered 232 genes that were candidate effectors(Supplemental Data Set 2) Because F virguliforme is a hemi-biotrophicpathogenwealsosearched theF virguliformegenomefor genes encoding carbohydrate active enzymes (CAZymes) anddiscovered 365 genes with potential functions in carbohydratemetabolism (Supplemental Data Set 3) In total these data setsprovided a resource to explore the transcriptomic variability ofF virguliforme across hosts

Fungal Infection by F virguliforme Produced Different RootPhenotypes on Maize versus Soybean

To understand transcriptome dynamics of F virguliforme inter-actions with maize (asymptomatic) versus soybean (symptom-atic) we profiled F virguliformendashinfected roots over a 2-week timecourse and collected samples for RNA-sequencing (RNA-seq)analysis A survey of early postinoculation time points allowed usto characterize the continuum of fungal attachment growthpenetration differentiation and symptom development By theend of the 2-week time course soybean roots showed signs ofnecrotrophy in both the tap and hypocotyl regions (Figure 2A)Additionally fungal-induced root necrosis had spread to de-veloping lateral roots adjoining the tap root This type of symptomdevelopment is consistent with root disease progression of SDSwhich begins as an asymptomatic biotrophic interaction with thefungus depending on living plant tissue but then turns ne-crotrophic with the fungus eventually killing host tissue In theasymptomatic host maize we did not observe any striking evi-denceof rootchlorosisornecrosisover the14-d timecourseof theexperiment (Figure 2A)Tomonitor in planta fungal growthweused trypanblue staining

to visualize fungal hyphae on both soybean and maize rootsthroughout the time course Although fungal growth and coloni-zation were apparent in both hosts the developmental stagevaried depending on the host plant (Figure 2B) For examplefollowing inoculation fungal spore germination was apparent onboth hosts and by 2 d after inoculation (DAI) fungal mycelia hadexpanded across the root surface Interestingly mycelia onmaizeroots grew parallel to root epidermis cells whereas myceliagrowth on soybean roots did not have any apparent directional

Figure 1 Syntenic Regions between Genome Versions of Fusariumvirguliforme

(A) Plot of syntenic regions retained between genome version 1 (v1) andgenome version 2 (v2) Diagonal lines including differences in lengthsillustrate distances of overlap between F virguliforme v1 and v2 genomeversions and the syntenic regionsbetween the scaffolds (Sca) and contigs(Ctg) in each(B) Micro-collinearity between scaffold 1 of genome v1 and contig 1 ofgenomev2connectedbyshadedgrayareasRegionscontaininggenesarehighlighted in greenor blue for forward or reverse orientation respectively

338 The Plant Cell

of PacBio data (Supplemental Figure 1) Filtered reads were as-sembled using the long-read optimized assembler Canu (Korenet al 2017) and resultant contigswere error-corrected using 503Illumina data using Pilon (Walker et al 2014) Our assembledFvirguliformegenomeencompassed52MBwith96contigswithanN50 of 154 MB (Supplemental Table 1) The resultant genome size(Mb) was slightly larger than the version 1 (v1) draft assembly(Srivastava et al 2014) and in this study the contiguity and N50were significantly improved (Supplemental Table 1) Syntenybetween the two genome versions was highly fragmented(Figure 1A) perhaps a result of having more than 3000 contigs inthe first version of the genome Notably within syntenic regionsmicro-collinearity between the two genomes was highly con-served (Figure 1B)

The F virguliforme genome generated in this study was an-notated using FunGAP (Min et al 2017) incorporating AUGUS-TUS MAKER and BRAKER gene model prediction algorithms

(Stanke et al 2006 Cantarel et al 2008 Hoff et al 2016) A totalof 16050 genes from the F virguliforme version 20 (v2) genomewere discovered representing an increase of 1205 genes com-pared with version v1 Comparisons of the coding sequencesbetween two genome versions revealed 12306 conserved geneswith aminimum 70gene alignment rate and 95 identity 1422genes were considered as misassembled or incomplete in one ofthe genomes Overall 2889 new genes that were missing in v1were annotated in v2 and were considered novel (SupplementalData Set 1) This gene set was enriched with genes involved inprotein ubiquitination organic compound breakdown and por-phyrin compound biosynthesis (Supplemental Table 2) Nextthe completeness of genome annotation was evaluated usingBenchmarking Universal Single-Copy Orthologs (Simatildeo et al2015 Waterhouse et al 2018) and we observed an 98completionwith10162of the16050genesbeing supportedwithprotein evidenceFurther explorationof thegenomeusingSignalP(v41 Petersen et al 2011) and EffectorP (v20 Sperschneideret al 2016) discovered 232 genes that were candidate effectors(Supplemental Data Set 2) Because F virguliforme is a hemi-biotrophicpathogenwealsosearched theF virguliformegenomefor genes encoding carbohydrate active enzymes (CAZymes) anddiscovered 365 genes with potential functions in carbohydratemetabolism (Supplemental Data Set 3) In total these data setsprovided a resource to explore the transcriptomic variability ofF virguliforme across hosts

Fungal Infection by F virguliforme Produced Different RootPhenotypes on Maize versus Soybean

To understand transcriptome dynamics of F virguliforme inter-actions with maize (asymptomatic) versus soybean (symptom-atic) we profiled F virguliformendashinfected roots over a 2-week timecourse and collected samples for RNA-sequencing (RNA-seq)analysis A survey of early postinoculation time points allowed usto characterize the continuum of fungal attachment growthpenetration differentiation and symptom development By theend of the 2-week time course soybean roots showed signs ofnecrotrophy in both the tap and hypocotyl regions (Figure 2A)Additionally fungal-induced root necrosis had spread to de-veloping lateral roots adjoining the tap root This type of symptomdevelopment is consistent with root disease progression of SDSwhich begins as an asymptomatic biotrophic interaction with thefungus depending on living plant tissue but then turns ne-crotrophic with the fungus eventually killing host tissue In theasymptomatic host maize we did not observe any striking evi-denceof rootchlorosisornecrosisover the14-d timecourseof theexperiment (Figure 2A)Tomonitor in planta fungal growthweused trypanblue staining

to visualize fungal hyphae on both soybean and maize rootsthroughout the time course Although fungal growth and coloni-zation were apparent in both hosts the developmental stagevaried depending on the host plant (Figure 2B) For examplefollowing inoculation fungal spore germination was apparent onboth hosts and by 2 d after inoculation (DAI) fungal mycelia hadexpanded across the root surface Interestingly mycelia onmaizeroots grew parallel to root epidermis cells whereas myceliagrowth on soybean roots did not have any apparent directional

Figure 1 Syntenic Regions between Genome Versions of Fusariumvirguliforme

(A) Plot of syntenic regions retained between genome version 1 (v1) andgenome version 2 (v2) Diagonal lines including differences in lengthsillustrate distances of overlap between F virguliforme v1 and v2 genomeversions and the syntenic regionsbetween the scaffolds (Sca) and contigs(Ctg) in each(B) Micro-collinearity between scaffold 1 of genome v1 and contig 1 ofgenomev2connectedbyshadedgrayareasRegionscontaininggenesarehighlighted in greenor blue for forward or reverse orientation respectively

338 The Plant Cell

pattern of colonization Also by 2DAI round and swollenmycelialstructures were observed on soybean roots and these structuresresemble penetration structures (eg appressoria) Support forthis classification comes from documented observations of in-fection pegs and appressoria development during in vitro Fvirguliforme infection of soybean radicals (Navi and Yang 2008)Interestingly these infection-like structures were also observedon maize but not until 7 DAI indicating a slower infectionprocess From 7 to 14 DAI we continued to record fungal growthand development at the site of inoculation and we observed anincrease in colonization by mycelia on both hosts By 14 DAIhowever masses of developing macroconidia were apparent onsoybean roots but not on maize roots indicating that asexualreproduction had initiated in the symptomatic host The transitionto necrotrophy was indicated by the induction of discoloration ofsoybean roots at 7DAI followed by necrosis at 10DAI (Figure 2B)Inmaize no visible symptomswere observed throughout the timecourse of the experiment

After we confirmed in planta growth of F virguliforme on bothsoybean and maize we conducted RNA-seq at six selected timeintervals over the course of the infection Additionally we alsocollected samples of F virguliforme macroconidia spores thatwere generated via in vitro germination After lower quality readsand adaptors were trimmed reads were mapped to the F vir-guliformegenomev2 Inour initial analysiswe identified low levelsof fungal mRNA reads representing only 004 to 013 of thetotal readsat0 to2DAI (Figure2C)While thiswasnotunexpectedwe generated aminimumof 200million reads per sample (at 0 to 4DAI) yielding read counts greater than 80000 per biologicalreplicate (Supplemental Figure 2 Supplemental Table 3) As

expected the percent ofmRNA reads aligning varied by host overthe time course Fungal reads frommaize (the asymptomatic host)increased in a linear fashion over the time course ranging from 111 to 253 of the total reads at 7 to 14 DAI However fungalreads from soybean (the symptomatic host) did not increasesubstantially until 7 DAI (Figure 2C) At this point the percent offungal reads approached those observed from F virguliformendashi-noculated maize samples In soybean this increase coincidedwith both symptom development and a concomitant shift frombiotrophy to necrotrophy

Host-Induced Gene Expression Profiles in F virguliforme

To determine whether the colonization profile of F virguliformediffered in a manner consistent with the differing host phenotypewe used a comparative transcriptomic-based approach Wehypothesized that this approachwouldbetter positionus todefinethe transcriptional reprogramming specific to each host Addi-tionally as a function of a single common pathogen interactionwe expected that this would also reveal the influence of the hostresponse on fungal gene expression First to determine whetherpathogen treatments were globally distinct from one another weperformed a principle coordinate analysis of all 39 samples frommaize and soybean colonization assays as well as samples fromin vitro assays of germinatingmacroconidia Using this approachwe observed that fungal responses were primarily correlated withtreatment (Supplemental Figure 3) and that the germinatingmacroconidia formed a distinct group from samples colonizinghosts While F virguliforme response on hosts did form a singlegroup all samples were distinctly separate within this group as

Figure 2 Fusarium virguliforme Pathogen Assays on Soybean and Maize

(A)Plant growth and development of soybean andmaize 14DAIwith F virguliforme Representative images showuninoculated (Mock) and F virguliforme-inoculated (Inoc) plants Bar 5 4 cm(B)Trypanblue stainingof inoculation siteson soybeancvSloan andmaize cvE13022S roots either inoculatedwithF virguliformeormock inoculatedBars5 16 mm Arrows point to areas of fungal growth and development asterisks highlight appressoria-like structures(C) The percentage of unique RNA-seq reads aligned to F virguliforme genome v2 Reads were trimmed by Trimmomatic v033 and aligned to Fusariumvirguliforme genome v2 with HISAT2 v210 Each sample is indicated by a colored dot and lines represent the mean of three biological replicates Grayshade indicates SEM

Fungal Transcriptional Plasticity Between Hosts 339

a function of host Intriguingly gene expression from both hostswere separated by time as well with the greatest separationidentified at time points between 4 and 14 DAI Additionallya separation from theplant sampleswasapparent in readsderivedfrom 7 to 14DAI samples of F virguliformendashinfected soybean Thegrouping of samples by hosts suggests plant-fungal interactionsgreatly shaped F virguliforme gene expression

To discover F virguliforme genes that were induced by hostinteraction we next compared gene expression patterns of Fvirguliforme on soybean or maize with in vitro germinated mac-roconidia Using this approach we identified 4192 and 4072unique F virguliforme genes that were differentially upregulated(log2 fold change gt 1) in fungal samples from soybean and maizerespectively throughout the time course As many genes were

highly induced we filtered the differentially expressed genes (log2

fold change gt 2)j to 3171 and 3010 genes to discover processesrelevant to soybean or maize colonization respectively Of thesignificantly upregulated genes from F virguliforme on soybeanthe vast majority of genes were induced at 0 7 10 and14 DAI(Figure 3A) Similarly themajority of induced F virguliformegeneswithinmaize roots were from samples derived at 7 10 and 14 DAI(Figure 3B) Surprisingly while the read depth was less in maizesamples at 0 DAI than soybean samples an additional 127 geneswere detected as significantly upregulated at log2 fold change gt 2highlighting an elevated response in F virguliforme to maizeversus soybean The greatest changes of unique gene upregu-lation occurred between 4 and 7DAI onmaize and at 10 to 14DAIon soybean (Supplemental Table 4) As a function of expression

A

C

B

D

Figure 3 Temporal Expression Patterns of F virguliforme Response Genes in Soybean and Maize Hosts versus in Germinating F virguliformeMacroconidia

(A) and (B) Number of differentially expressed (DE log2(FC) gt 2) F virguliforme genes in roots of soybean (A) or maize (B) hosts versus geminatingF virguliforme macroconidia(C) and (D) Heat maps of significant (log2(FC) gt 2) enrichment of gene ontology categories of upregulated F virguliforme genes across pooled time pointsduring F virguliforme colonization of soybean (n 5 233 [C]) and maize (n 5 165 [D])

340 The Plant Cell

between both hosts and comparison of host differential geneexpression between time points we observed that only threegeneswere conserved Of these three genes one (Fvm1_12746)was functionally annotated as phosphonopyruvate decar-boxylase a component of organic acid production in fungi(Yang et al 2015a)

Weuseda functional geneontology (GO)enrichment analysis toexplore the functionofhost inducedgenes inF virguliformeOf themore than 50 biological process categories that were enrichedseveral processeswere consistently upregulated in both soybeanand maize including carboxylic acid lipid or cofactor bio-synthesis as well as polysaccharide metabolism protein de-phosphorylation and small-molecule biosynthesis (Figures 3Cand 3D Supplemental Data Sets 4 and 5) Overall expressionpatterns on both hosts were similar for lipid and cofactor bio-synthesis which is not surprising as these processes are criticalfor fungal growth and signaling pathways (Schrettl et al 2007Lysoslashe et al 2008) Carboxylic acid biosynthesis was inducedthroughout the colonization time course andwe hypothesize thatthis is a critical process for secondary metabolite production insupport of fungal colonization regardless of the host (Brown andProctor 2016) Interestingly protein dephosphorylation and smallmolecule biosynthesis were enriched in fungal transcriptomicprofiles andwere elevated in expressionwhen colonized soybeanroots at 7 to 10 DAI

Temporal Divergence of Gene Coexpression Upon HostColonization by F virguliforme

As noted above specific genes related to processes associatedwith F virguliforme colonization and development were differ-entially inducedatdistinct temporal stagesduring the timecourseTo extend our investigation we were interested in the divergenceof global gene coexpression patterns during colonization of bothmaize and soybean Differential coexpression patterns couldreflect F virguliforme transcriptomic dynamics stemming frombiotrophic to necrotrophic transitions during infection To addressthis we applied a weighted gene correlation network analysis onthe F virguliforme expression data collected from maize andsoybean rootsExpressiondatasetswerefiltered to removegeneswith low expression before construction of the coexpressionnetwork leaving 11112 genes after filtering Next we built in-dividual networks formaizeandsoybeanRNA-seqdata andthesegenes were clustered into 22 modules for F virguliforme coloni-zation of maize and 20 modules for F virguliforme colonization ofsoybean (Supplemental Figures 4 and 5 Supplemental Data Sets6 and 7) Then we assigned the modules to four large groupsbased on their temporal patterns (1) early induced expression at 2DAI but downregulated at 4 DAI (2) elevated expression at 4ndash7DAI (3) induced expression at 7ndash10DAI and (4) downregulation ofexpression from 2 to 4 DAI but induced at 10 DAI (Figure 4) Whilethese temporal patterns of coexpression modules across F vir-guliforme colonization appear similar when placed within thesefour large groups the gene enrichment of modules containedwithin these groups varied significantly by host

Gene coexpressing modules in Group 1 from the F virguli-formendashmaize interaction network were enriched for putativenegative regulatory elements for many functional processes

including cellular metabolism macromolecule production andexpressionof primarymetabolism (Supplemental DataSet 6) Thisindicates that F virguliforme was repressing secondary metab-olism and using self-derived energy at early interaction times withmaize roots However processes upregulated in F virguliformecolonizing soybean roots at 2 DAI were enriched for reactiveoxygen species (ROS) generation and oxalic acid production(Supplemental Data Set 7) Both of these processes are associ-ated with early hemibiotrophic and necrotrophic plant fungal in-teractions at early time points in colonization ROS in fungalhyphae supports the differentiation of cells for infection structureslike appressoria (Heller and Tudzynski 2011) We observed thedevelopment of appressoria like structures at 2 DAI in soybeanroots (Figure 2B) suggesting that F virguliforme is already pen-etrating host tissues within 48 h of contact Oxalic acid bio-synthesis genes were also enriched in the assembled modulesindicating a potential downregulation of host cell death by au-tophagy in order to prevent a massive necrotic response by thehost killing the fungus similar to what was seen with Sclerotiniasclerotium (Veloso and van Kan 2018) Taken together theseanalyses suggest that F virguliforme infects and manipulates thesoybean host responses as early as 2 DAINumerous coexpression modules were upregulated at 4ndash7 DAI

in both maize- and soybean-inoculated with F virguliforme

Figure 4 Distinct Gene Coexpression Groups of Host-Induced Fusariumvirguliforme Response Genes

(A) and (B) Temporal expression profile of F virguliforme gene coex-pression modules during colonization of maize (A) or soybean (B) rootsacross the timecourseModulesweregrouped into fourdistinctexpressionpatterns (1) upregulation at 2 DAI (2) upregulation at 4ndash7DAI (3) inductionat 7ndash10 DAI and (4) increase in expression at 10ndash14 DAI For each timepoint the averaged expression of genes contained within a given modulewas plotted

Fungal Transcriptional Plasticity Between Hosts 341

Interestingly most of these modules were also highly expressedfor the remainder of the colonization time course (Figure 4ASupplemental Figure 4) and were further enriched for processesassociated with primary metabolism similar to Group 1 (Table 1)However processes associated with response to the host de-fenses were also enriched For example at 4 DAI carboxylic acidbiosynthesis-associated genes and related processes were up-regulated suggesting that toxin production was occurring(Supplemental Data Set 6) Conversely the same processes fromF virguliforme within soybean highlighted a faster colonizationprogram Enrichment of processes associated with cellular cat-abolic processes of cellulose pectin and polysaccharides in thesoybean infection samples were identified Of interest and rele-vance to the host-association and symptomatic nature of the Fvirguliformendashsoybean interaction we also observed an enrich-ment at 4 DAI in small molecule biosynthesis including thosepotentially associated with the function of necrotrophic effectors(Supplemental Data Set 7 Chang et al 2016a) In total theseobservations highlight the initial transition from a biotrophicto necrotrophic lifestyle coincident with the modification andbreakdown of host tissue to enable further proliferation (Laluk andMengiste 2010)

One striking observation from this analysis is that numerousdiverse processes were enriched in modules upregulated at 7ndash10DAI inF virguliformendashmaizesamplesOf these theupregulationofNADP stood out as this process has been previously associatedwith hyphal differentiation initiation for infection structures (Hellerand Tudzynski 2011) This is consistent with our phenotypicobservations shown in Figure 2B at 7 DAI The upregulation ofgene processes in F virguliforme associated with catabolism ofamino acid sugars also suggests access to plant-derived com-pounds likely via direct penetration of the host tissue by thefungus Concomitant with this upregulation of processes asso-ciated with chemical stimulus likely indicates F virguliforme wassensing host defense response involving the production andsecretion of antimicrobial compounds During this same timeframe while F virguliforme from maize was activating nutri-ent access-associated processes F virguliforme upregulatedprotection-associated mechanisms in soybean including anti-biotic catabolism response toROS andchemical to host defenseactivation

By 14 DAI processes associated with Group 4 (Figure 4) wereexpressed as a function of host colonization For example pro-cesses involving primarily amino acid sugar and nitrate acqui-sition were induced in samples derived from maize However atthe same time point in samples from soybean necrotrophicprocesses had initiated with an enrichment in functions associ-ated with cell killing organic acid transport and self-protectionfromhost inducedROSbycell redoxhomeostasis Theseprocessenrichments were supported by the observed necrotrophic en-velopment of the soybean tap root at 14 DAI (Figure 2B)The lack of temporal conservation of enriched processes be-

tween colonization of these two hosts highlights plasticity of theF virguliforme transcriptomeOverall fewgeneswerecoexpressedinasimilarmannerwithinmoduleswhencomparedbetween thesetwo hosts (Figure 5) Moreover only 8of genes were conservedbetween coexpression networks of F virguliformendashinoculatedsoybean and maize Comparison of gene overlap highlights thatprocesses enriched in group 3 of coexpression in maize containmoregenes fromthe temporal ofF virguliformeacrossall soybeancoexpression groups Interestingly Module 14 in Group 2 ofF virguliformewithin soybean contained the greatest overlapwithseveral early (2 to 4 DAI) induced maize modules Module 14 wasthe largest coexpression module with 3503 genes and it maycontainmanygenes relevant tobasic cellular functionsneeded forviability and growth (Supplemental Figure 5 Supplemental DataSet 7)

Host-Specific Gene Expression Patterns DuringRoot Colonization

As noted above we observed a temporal divergence of biologicalprocesses enriched by respective host colonization To furtherexplore thiswenext asked if these induction responseswerehostspecific Previous work comparing the infection profiles of phy-topathogenic Zymoseptoria tritici on wheat revealed temporalvariation of isolate infection (Haueisen et al 2018) To determinewhether this is also the case in F virguliforme we directly com-paredF virguliforme gene expression fromeachhost at each timepoint (Figure 6A) The majority of genes (81 9002) in theF virguliforme transcriptome were not differentially regulated incross-species colonization Of the proportion of genes that were

Table 1 Selected Gene Ontology Enrichment of Distinct Gene Coexpression Groups Identifies Manipulation of the Fusarium virguliformeTranscriptome for Host Colonization

Grouping

Host

Maize Soybean

1 Negative regulation of biological processes ROSOxalic acid production

2 Primary metabolism Catabolic processes of celluloseDefense to host Small molecule biosynthesis

3 Amino acid sugar catabolism Antibiotic catabolic processOxidation-reduction process Response to ROS

4 Nitrate transport Killing of cells of other organismAmino acid sugar catabolism Cell redox homeostasis

A full list of coexpression module gene ontology enrichment within formulated groups is provided in Supplemental Data Sets 6 and 7

342 The Plant Cell

differentially induced43(924genes)wereuniquelyupregulatedduring maize root colonization 56 (1186 genes) were uniquelyupregulated upon colonization of soybean and 01 wereconsistently upregulated at multiple time points during the in-fection time courseOf these differentially inducedgenes the vastmajority were induced at 10 and 14 DAI (Supplemental Table 5Supplemental Data Set 8) While fewer genes were differentiallyupregulated at early time points these genes highlight specificprocesses underlying temporally distinct stages of fungal colo-nization Interestingly genes highly upregulated (log2 foldchange gt 20) at 0 DAI within F virguliforme colonizing soybeanroots were related to DNA methylation suggesting that thisprocess was induced by host signals in soybean roots No bi-ological processes were uniquely enriched during maize rootcolonization at 0DAI It is likelyF virguliforme began to respond tohost-induced antimicrobial metabolites at 2 DAI by upregulatingABC transporters (Gupta and Chattoo 2008) and by initializingtoxin secretion using terpene synthases as the fungus grew in themaize roots Based on the unique set of upregulated genes duringsoybean colonization at 2 DAI F virguliforme was penetratingroots via ROS production and upregulation of Zn(II)-Cys6 fungaltranscription factors (Brown et al 2007)

F virguliforme colonization of soybean roots resulted in theactivation of marked fungal defense signals at 4 DAI as in-dicatedby the rapid andstrong induction (log2 fold changegt10) ofvarious cytochrome oxidase genes Interestingly these genes

were not upregulated at the same time in samples derived fromF virguliformendashmaize colonization suggesting that the fungus hadnot penetrated the root andor a lack of antimicrobial metaboliteaccumulation However at 7 DAI cytochrome oxidases andNADP was upregulated in F virguliformemaize interactions thusindicating cellular differentiation of hyphal penetration structures(Heller and Tudzynski 2011) At the same time cellular degra-dation and nutrient access-associated processes were signifi-cantlyupregulated (log2 foldchangegt10) inFvirguliformendashcolonizedsoybean samples as indicated by the expression of glycosidehydrolases and pectinases as well as various nutrient trans-porters A larger set of genes was differentially induced in Fvirguliforme between hosts at 10 and 14 DAI At these timepoints samples from maize revealed an enrichment of genesassociated with secretion and catabolic processes (SupplementalDataSet 9) while those fromsoybean revealed a shift to processesindicative of fungal growth (eg glycerolipid and lipoprotein bio-synthesis Takahashi et al 2009) This is in support of our obser-vation of asexual production at 14 DAI upon soybean roots(ie Figure 2B)Central to defoliation of the host during F virguliforme infection

is the secretion of proteinaceous phytotoxins produced by thepathogen resulting in host foliar SDS symptom development(Chang et al 2016a) In this study genes involved in toxin andsecreted protein production were induced in a time-dependentmanner aswere their levelsofexpressionwhencomparedacrosshosts (Figures 6B and 6C) Moreover while nearly triple thenumberofpredictedeffector-encodinggeneswereupregulatedat0ndash2 DAI in soybean none of the candidate effector-encodinggenes were differentially induced between hosts at these timepoints By 4 DAI almost four times as many candidate effectorswere upregulated in soybean roots (Supplemental Figure 6) in-cluding those with predicted functional domains associated withpectin lyases glycoside hydrolases and necrosis-inducing pro-teins However similar to the above patterns all but three geneswere not considereddifferentially expressedwhencomparedwithF virguliforme colonization of maize (Supplemental Data Set 10)Until this point in the infection process candidate CAZymesexpression profiles were induced in similar patterns in the twohosts and no differentially expressed CAZymes genes were de-tected at 0 2 and 4 DAI (Supplemental Figure 7) However at 4DAI pectin lyases and glycoside hydrolases were expressed atmuch higher levels (gt10 log2) by F virguliforme in soybean roots(Supplemental Data Set 11) This trend was exacerbated at 7 DAIwith numerous CAZymes and effectors related to pectin lyasesbeing upregulated in soybean roots colonized by F virguliformeThis suggests divergence in fungal colonization programs be-tweenmaize and soybean at 7 DAI potentially stemming from theshift of biotrophic to necrotrophic as visible symptoms started toinitialize at 7DAI A necrotrophic lifestylewas evident by 10 and14DAIwith 14and28candidate effectors and37and114candidateCAZymes respectively targeting cellular breakdown of soybeanroots by F virguliforme Conversely few pectin lyases were ex-pressed during F virguliforme colonization of maize roots Weposit that reduced expression of lyases may stem from the basicphysiological differences between maize and soybean Indeedmonocot roots contain only 10 pectin whereas eudicotscontain up to 30 (Caffall and Mohnen 2009) The effectors that

Figure 5 Symptomatic and Asymptomatic Hosts Reveal TranscriptomicPlasticity during F virguliforme Colonization

The percent of overlapping genes from the weighted gene correlationnetwork analysis modules was calculated as the ratio of the number ofshared genes between F virguliforme expression on each host divided bythe total number of genes within the module that contained the fewestnumber of genes Modules are annotated with grouping assignments asshown in Figure 4 The increasing shade of blue represents increasingpercent module overlap of the gene count shared between the twomodules

Fungal Transcriptional Plasticity Between Hosts 343

were uniquely induced between 7 and 10 DAI in maize roots arecurrently putative effectors with no known function

DISCUSSION

Comparative systems-based approaches using pathogenic andnonpathogenic fungal isolates have allowed us to identify geneticsignatures associated with pathogenicity and compatible host-pathogen interactions With the use of single-pathogensingle-host approaches a more complete understanding of the con-tinuum of pathogenic and endophytic niches determining hostrange is emerging We used a high-resolution transcriptome-based approach to define how a single fungal pathogen can re-wire infection processes and host defense programs to promoteeither symptomatic or asymptomatic colonization depending onthe host To accomplish this we assembled and annotated theF virguliforme genome de novo generated 39 mRNA tran-scriptomes across in vitro and in planta time courses to identifyinfection program modulation across two hostsmdasha symptomaticsoybean host and an asymptomatic maize host We found

distinct changes in root phenotypes as a function of host duringF virguliforme colonization For example maize roots remainasymptomatic whereas soybean roots turn chlorotic and even-tually become necrotic Underlying these phenotypic distinctionsare myriad of host-dependent transcriptional programsIn this study we observed that temporal changes in tran-

scriptional dynamics during F virguliforme colonization of maizeroots were largely gradual over the infection time course Con-versely F virguliforme colonization of soybean caused dramaticchanges in transcriptional activity at 4ndash10 DAI as exhibited bycoexpression module gene enrichment This is illustrated by ourobservation of signaling associatedwith small molecule secretionand host cell death processes These observed changes areconsistent with previously described transcriptional dynamics ofhemibiotrophic plant pathogens (OrsquoConnell et al 2012) For ex-ample small molecules secreted by plant pathogens are hy-pothesized to target host defensemachinery andor processes tomodulation immune responses to the fungus (Jennings et al2000 Chang et al 2016a) In parallel dephosphorylation of plantplasma membrane-localized proteins by fungal pathogens is

Figure 6 Unique Host Genes Induced within Fusarium virguliforme Highlight Disease Development

(A)Heatmap of expression profiles of significantly upregulated genes (log2(FC) gt 1) at a single time point in F virguliforme colonizing soybean ormaize (n52099) Yellow color indicates differentially upregulated genes from F virguliforme colonization of maize and gray color indicates differentially upregulatedgenes from F virguliforme colonization of soybean(B) Expression patterns of significantly upregulated candidate effector genes at a single time point in maize or soybean over the infection time course Thecolored lines indicate the means of all genes in the plot Gray lines represent individual genes(C)Expressionpatternsof significantly upregulatedcandidatecarbohydrateactiveenzymesatasingle timepoint inmaizeor soybeanover the infection timecourse The colored lines indicate the means of all genes in the plot Gray lines represent individual genes

344 The Plant Cell

regarded as a critical process to prevent signaling cascades thatnormally stimulate host defense responses (Yang et al 2015b) Intotal the upregulation of these processes in F virguliforme duringsoybean colonization suggests an infection strategy to reducehost immune responses more so than when F virguliformecolonizes maize roots

Interestingly the identified differences in transcriptomes doesnot appear to be a result of unique gene expression by each hostbut rather is a result of the temporal induction of genes with re-spect to the degree of host colonization In support of this genecoexpression networks highlighted the temporal processesunique to each host through varying stages of fungal growthinfection and proliferation each of which coincidedwith changesin pathogen access to nutrient sources The rapid growth andinfection of F virguliforme on soybean roots by 2 DAI indicatesa rapid recognition of the host surface and initiation of the earlyinfection program (Elliott 2016) However F virguliforme geneexpression patterns on maize roots through the early time courseof infection were enriched for negative regulation of biologicalprocesses and primary metabolism suggesting that this funguswas not immediately stimulated to infect the maize host Upre-gulation of processes indicative of maize infection did not oc-cur until 7 DAI illustrating a delay in host-fungal recognition(Giovannetti et al 1994) In soybean upregulation of recognition-associated processes occurred at 2 DAI

By the timeF virguliforme hadpenetratedmaize roots (7DAI)infection on soybean had already begun to transition from a bio-trophic lifestyle to a necrotrophic infection Upregulation of smallprotein secretion and fungal-derived toxin production demon-strates host cell modification by F virguliforme in soybean rootskey events associated with and required for nutrient access(Sahu et al 2017) Throughout the remainder of the time course ofinfection we observed a general increase in the gene expressionassociated with cell death and pathogenicity in F virguliformendashinfected soybean Conversely in maize we observed the ex-pression of fewer catabolic process-associated genes indicatinga general reduction in processes associated with nutrient ac-quisition Based on these observations we surmise that thecomparative analysis of the interaction of F virguliformewith twohosts supports our hypothesis of a divergence in the transcriptomeof this fungus Indeed while the vast majority of the transcriptomewas expressed during fungal colonization of both maize and soy-bean the induction of genes underlying distinct often divergentbiological processes were temporally distinct This observationagrees with previous studies which identified temporal changes ofgene expression during colonization of hosts by the same fungusexhibiting different lifestyles (Lahrmann et al 2013 Lorrain et al2018) and this may suggest a reduction in a shift to necrotrophyon maize

Because transcriptome expression of F virguliforme variedduring colonization of soybean versus maize our study offersa unique perspective to identify processes critical for necrotrophyon soybean For example previous analyses concluded that theupregulation of genes at 4 DAI that encode effectors and CA-Zymes highlights the start of the transition from biotrophy tonecrotrophy (Changet al 2016aChanget al 2016bNgaki et al2016) In the current study we also observed an increase in theexpressionofCAZyme-andpectin lyase-associated transcripts in

soybean compared with maize Moreover expression of theNecrosis InducingProteinwashighly upregulated during soybeancolonization over the time course of analysis suggesting a pos-sible dicot-specific response as previously hypothesized by Baeet al (2006) Indeed fungal effectors and CAZymes were ex-pressed in temporally distinct waves at 2 4 and 7DAI in soybean-infected roots yet not inmaizeMoreover eachwave increased inintensity of gene expression In total these expression patternsagree with previous data further supporting the hypothesis thatthe upregulation of cell-degrading and necrosis-inducing pep-tides is a key step in the shift from biotrophy to necrotrophy(Kleemann et al 2012 Haueisen et al 2018)While a hemibiotrophic infection program ensued in soybean

infection was delayed and catabolic activities as inferred bytranscriptional analysiswere lowerwhenF virguliformecolonizedmaize Either a lack of host recognition by F virguliforme(Giovannetti et al 1994) or an upregulation of host defenses frompattern triggered immunity (Bagnaresi et al 2012 Zhang et al2018) would slow fungal growth and downregulate developmentOnce inside the host fewer effectors andCAZymeswere uniquelyexpressed in maize roots and were more often than not down-regulated after induction In total the lower level of expressionalong with the decrease in CAZyme diversity suggests that thecellular environment within maize roots did not stimulate prolificupregulation of necrosis-inducing peptides This may stem fromthe physiological differences in cell structure between monocotsanddicots (Caffall andMohnen 2009) Additionally as theprimaryhosts forF virguliforme are legumesF virguliformemaynot be asadapted to colonize monocots (Zhao et al 2013)The full understanding of how processes that are required for

and regulate the transition from biotrophy to necrotrophy duringthe hemibiotrophic stage of fungal growth are regulated hasremained elusive (Rai and Agarkar 2016 Chowdhury et al 2017Haueisen et al 2018) However it is hypothesized that tran-scription factors likely play a critical role in these transitions in-cluding recent work from several groups that has shown thatmembers of the Zn(II)-Cys6 and C2H2 zinc-finger family of tran-scription factors alter pathogenicity andgrowth (Chen et al 2017Sang et al 2019) In this study we found that several Zn(II)-Cys6genes were uniquely upregulated during early soybean coloni-zation a process we hypothesize may lead to an enhancement ofpathogenicity of F virguliforme on soybean Our current studypoints to several key processes (eg transcriptional regulation ofpectin lyase biosynthesis genes and fungal toxin biosynthesisgenes) that are specifically associated with the lifestyle transitionfrom biotrophy to necrotrophy in association with soybeanConversely these same transcriptional transitions were signifi-cantly reduced in F virguliforme when associated with theasymptomatic host maizeWe propose that these gene expression profiles highlight the

transcriptional plasticity of a single fungal isolate on multiplehosts In this regard the analysis highlights the significance ofrewiring during host-pathogen interactions including the tem-poral expression of distinct gene networks underpinning thedevelopment of asymptomatic and symptomatic programsWhileadditional work remains this study illustrates the significance oftwo distinct niches within agroecosystems of this importantpathogen (Rai and Agarkar 2016) one niche that supports the

Fungal Transcriptional Plasticity Between Hosts 345

maintenance and survival of a pathogen in the absence ofa compatible host and another that supports proliferation andspread of a pathogen resulting in significant yield losses of animportant staple crop The ability of F virguliforme to function inthese two distinct roles suggests the need to consider the ge-nomic potential and ability of plant pathogens to express a gra-dation of transcriptional programs which in turn imparts lifestyleplasticity on a broad range of hosts

METHODS

Genome Sequencing Assembly and Annotation forFusarium virguliforme

PacBio and Illumina sequencing were performed using high molecularweightDNAextracted from lyophilized (FreeZone25 Labconco)Fusariumvirguliforme Mont-1 mycelia grown for 4 weeks in potato dextrose broth(Millipore-Sigma catalog no P6685) The F virguliforme Mont-1 isolatehas been extensively used as a model for the advancement of our un-derstandingof soybean (Glycinemax)SDS includingservingasamodel forgenomics and transcriptomics (Srivastava et al 2014 Ngaki et al 2016Sahuet al 2017) for theanalysis of pathogeneffector biology (Changet al2016a Chang et al 2016b) DNA was extracted using a modified cetyltrimethylammonium bromide procedure with 1 polyvinylpyrrolidone(Lade et al 2014) A PacBio library was constructed at the University ofGeorgiaGenomicsandBioinformaticsCore andsizeselected for 15- to20-kb fragmentsusing theBluePippin system (SageScientific) The librarywassequencedonaSequelPlatform and thesingle smart cell yielded65Gbofread data

For error correction Illumina TruSeq Nano DNA libraries were preparedand sequenced on an Illumina MiSeq v3 for a lane of 23 300 nucleotidesand HiSeq 4000 for a lane of 2 3 150 nucleotides at Michigan StateUniversity Research Technology Support Facility PacBio reads wereassembled and error corrected using Canu (v18 Koren et al 2017) usingdefault parameters with several modification including minReadLength5

2000 GenomeSize 5 51Mb minOverlapLength 5 1000 A default maxerror rate of 024 was used for alignment during error correction and 0045foroverlapandassemblyThemax target coveragewassetat403 Ak-mersizeof16wasused foroverlappingduringerror correction andak-mer sizeof 22 was used for overlapping during assembly

The genome size estimate for assembly was extrapolated from theprevious F virguliformeMont-1 draft genome of 509Mb (Srivastava et al2014) Due to the presence of contaminating bacterial DNA in the initialassembly draft contigs were compared with the published F virguliformegenome assembly with LAST (v912 Kiełbasa et al 2011) and novelcontigs were validated for fungal origin by BLAST1 (v2230 Camachoet al 2009) against the nonredundant National Center for BiotechnologyInformation (NCBI) database The genome graph structure was visualizedin Bandage (httpsacademicoupcombioinformaticsarticle31203350196114 Wick et al 2015) to survey contiguity and ambiguities(Supplemental Figure 1) Assembled contigs were error-corrected usingPilon (v122 Walker et al 2014) and default settings using a total of 503coverage of Illumina paired-end 300 nt and 150 nt data for F virguliformePaired end reads were adaptor and quality trimmed using Trimmomatic (v033 Bolger et al 2014) and then were aligned to the draft contigs usingBowtie2 (v226 Langmead and Salzberg 2012) with default settings Pilonwas run five times sequentially until limited (ie lt100) corrections werefound The new genome assembly was compared to the previous genomeassembly by QUAST (v30 Gurevich et al 2013) and is referred to asF virguliforme genome v2

Transcript evidence for gene predictions was acquired from both theNCBI (Short Read Archive [SRA] SRR1382101) and germinating

macroconidia from the F virguliforme RNA-seq time course (see below)Readswereadaptor andquality trimmedusingTrimmomatic (v033Bolgeret al 2014) for all transcript evidence These reads were then analyzedusing Breaker MAKER and Augustus gene model prediction algorithmshoused within FunGAP (v10 Min et al 2017) as transcript evidence Theparameters for running FunGAP were set as ndashsister_proteome Fusar-iumndashaugustus_species fusarium_graminearum with transcript readsprovided asndashtrans_read_single The resulting annotation from FunGAPconsisted of 16050 genes Single-copy fungal orthologs from Bench-marking Universal Single-Copy Orthologs (v3 Simatildeo et al 2015) wereused to assess the completeness of the genome annotation

Functional annotation was completed using Trinotate (v311 Bryantet al 2017) Trinotate-annotated gene models with evidence from severaldatabases (NCBI nonredundant protein database Swissprot-Uniprotdatabase GO and InterpoScan) with BlastX finding single hit at anE-value thresholdof 1E25 (Altschul et al 1990)Weused this information topredict protein domains with HMMER (v31 Finn et al 2011) trans-membrane proteins with TMHMM (v20 Krogh et al 2001) rRNA withRNAmmer (v12 Lagesen et al 2007) secreted proteins with SignalP(v41 Petersen et al 2011) and GO with GOseq (Young et al 2010)Additionally EffectorP (v20 Sperschneider et al 2016) was used topredict fungal effectorswithin the secreted proteins and dbCAN (Yin et al2012) was used to identify F virguliforme CAZymes and secondarymetabolism genes

Comparative Genomics with F virguliforme

MCSCAN toolkit (v11 Wang et al 2012) was used to identify syntenicgenepairs between the second version (v2) and the first version (v1) of theF virguliforme genome Conserved gene blocks were discovered throughLAST alignment Plots of macro- and micro-synteny were created by theMCScan in python

To discover novel and retained genes the v2 F virguliforme genomewas compared to the v1 F virguliforme genome Coding sequences forgenemodelswere extracted from the v1 genomeby gffread (Trapnell et al2010) Next gene sequences were reciprocally compared by BLASTn(v2226 Altschul et al 1990) Genes with an e-value below 1E25 greaterthan 70 gene alignment and 95 gene identity were classified as re-tainedgenes If a genewas alignedwith 95 identitywith an e-valuebelow1E25 but less than 70 gene alignment it was denoted as a mis-assembled gene Genes that were annotated in v2 that did not have analignment to the v1 genome were considered novel

Plant and F virguliforme Assays

SoybeancvSloan (providedbyMartinChilversMichiganStateUniversity)and maize (Zea mays) hybrid E13022S (Epley Brothers Hybrids Inc pro-videdbyMartinChilvers)weresurfacesterilized for30s in70(vv) ethanolfor 30 s and 10 (vv) bleach for 20 min and then triple rinsed in steriledistilled water for 1 min After sterilization soybean seeds were placedinside a Petri dish containing two sheets of sterile 100-mmWhatman filterpapersoaked in5mLofsterilewaterSoybeanseedswere incubated for5din total darkness at 21degC to enable germination Maize seeds were in-cubated insterilewater for 24h indarknessandplacedbetween twosheetsof sterile 100 mm Whatman filter paper with 5 mL of sterile water insidea Petri dish Seeds were incubated for 5 d in total darkness at 21degC toensure germination

F virguliformeMont-1 was propagated on potato dextrose agar (Difco)for 7weeks Spores of asexualmacroconidiawere collected diluted to 105

macroconidiamL21 in sterilewater and sprayed onto germinatedmaize orsoybean seedlings with an elongated a radicle using a 3-oz travel spraybottle Twenty-fivesprayswereapplied to theseedlingsatanglesof0deg90deg180deg and 270deg to that ensure seedswere thoroughly inoculated Formock

346 The Plant Cell

regarded as a critical process to prevent signaling cascades thatnormally stimulate host defense responses (Yang et al 2015b) Intotal the upregulation of these processes in F virguliforme duringsoybean colonization suggests an infection strategy to reducehost immune responses more so than when F virguliformecolonizes maize roots

Interestingly the identified differences in transcriptomes doesnot appear to be a result of unique gene expression by each hostbut rather is a result of the temporal induction of genes with re-spect to the degree of host colonization In support of this genecoexpression networks highlighted the temporal processesunique to each host through varying stages of fungal growthinfection and proliferation each of which coincidedwith changesin pathogen access to nutrient sources The rapid growth andinfection of F virguliforme on soybean roots by 2 DAI indicatesa rapid recognition of the host surface and initiation of the earlyinfection program (Elliott 2016) However F virguliforme geneexpression patterns on maize roots through the early time courseof infection were enriched for negative regulation of biologicalprocesses and primary metabolism suggesting that this funguswas not immediately stimulated to infect the maize host Upre-gulation of processes indicative of maize infection did not oc-cur until 7 DAI illustrating a delay in host-fungal recognition(Giovannetti et al 1994) In soybean upregulation of recognition-associated processes occurred at 2 DAI

By the timeF virguliforme hadpenetratedmaize roots (7DAI)infection on soybean had already begun to transition from a bio-trophic lifestyle to a necrotrophic infection Upregulation of smallprotein secretion and fungal-derived toxin production demon-strates host cell modification by F virguliforme in soybean rootskey events associated with and required for nutrient access(Sahu et al 2017) Throughout the remainder of the time course ofinfection we observed a general increase in the gene expressionassociated with cell death and pathogenicity in F virguliformendashinfected soybean Conversely in maize we observed the ex-pression of fewer catabolic process-associated genes indicatinga general reduction in processes associated with nutrient ac-quisition Based on these observations we surmise that thecomparative analysis of the interaction of F virguliformewith twohosts supports our hypothesis of a divergence in the transcriptomeof this fungus Indeed while the vast majority of the transcriptomewas expressed during fungal colonization of both maize and soy-bean the induction of genes underlying distinct often divergentbiological processes were temporally distinct This observationagrees with previous studies which identified temporal changes ofgene expression during colonization of hosts by the same fungusexhibiting different lifestyles (Lahrmann et al 2013 Lorrain et al2018) and this may suggest a reduction in a shift to necrotrophyon maize

Because transcriptome expression of F virguliforme variedduring colonization of soybean versus maize our study offersa unique perspective to identify processes critical for necrotrophyon soybean For example previous analyses concluded that theupregulation of genes at 4 DAI that encode effectors and CA-Zymes highlights the start of the transition from biotrophy tonecrotrophy (Changet al 2016aChanget al 2016bNgaki et al2016) In the current study we also observed an increase in theexpressionofCAZyme-andpectin lyase-associated transcripts in

soybean compared with maize Moreover expression of theNecrosis InducingProteinwashighly upregulated during soybeancolonization over the time course of analysis suggesting a pos-sible dicot-specific response as previously hypothesized by Baeet al (2006) Indeed fungal effectors and CAZymes were ex-pressed in temporally distinct waves at 2 4 and 7DAI in soybean-infected roots yet not inmaizeMoreover eachwave increased inintensity of gene expression In total these expression patternsagree with previous data further supporting the hypothesis thatthe upregulation of cell-degrading and necrosis-inducing pep-tides is a key step in the shift from biotrophy to necrotrophy(Kleemann et al 2012 Haueisen et al 2018)While a hemibiotrophic infection program ensued in soybean

infection was delayed and catabolic activities as inferred bytranscriptional analysiswere lowerwhenF virguliformecolonizedmaize Either a lack of host recognition by F virguliforme(Giovannetti et al 1994) or an upregulation of host defenses frompattern triggered immunity (Bagnaresi et al 2012 Zhang et al2018) would slow fungal growth and downregulate developmentOnce inside the host fewer effectors andCAZymeswere uniquelyexpressed in maize roots and were more often than not down-regulated after induction In total the lower level of expressionalong with the decrease in CAZyme diversity suggests that thecellular environment within maize roots did not stimulate prolificupregulation of necrosis-inducing peptides This may stem fromthe physiological differences in cell structure between monocotsanddicots (Caffall andMohnen 2009) Additionally as theprimaryhosts forF virguliforme are legumesF virguliformemaynot be asadapted to colonize monocots (Zhao et al 2013)The full understanding of how processes that are required for

and regulate the transition from biotrophy to necrotrophy duringthe hemibiotrophic stage of fungal growth are regulated hasremained elusive (Rai and Agarkar 2016 Chowdhury et al 2017Haueisen et al 2018) However it is hypothesized that tran-scription factors likely play a critical role in these transitions in-cluding recent work from several groups that has shown thatmembers of the Zn(II)-Cys6 and C2H2 zinc-finger family of tran-scription factors alter pathogenicity andgrowth (Chen et al 2017Sang et al 2019) In this study we found that several Zn(II)-Cys6genes were uniquely upregulated during early soybean coloni-zation a process we hypothesize may lead to an enhancement ofpathogenicity of F virguliforme on soybean Our current studypoints to several key processes (eg transcriptional regulation ofpectin lyase biosynthesis genes and fungal toxin biosynthesisgenes) that are specifically associated with the lifestyle transitionfrom biotrophy to necrotrophy in association with soybeanConversely these same transcriptional transitions were signifi-cantly reduced in F virguliforme when associated with theasymptomatic host maizeWe propose that these gene expression profiles highlight the

transcriptional plasticity of a single fungal isolate on multiplehosts In this regard the analysis highlights the significance ofrewiring during host-pathogen interactions including the tem-poral expression of distinct gene networks underpinning thedevelopment of asymptomatic and symptomatic programsWhileadditional work remains this study illustrates the significance oftwo distinct niches within agroecosystems of this importantpathogen (Rai and Agarkar 2016) one niche that supports the

Fungal Transcriptional Plasticity Between Hosts 345

maintenance and survival of a pathogen in the absence ofa compatible host and another that supports proliferation andspread of a pathogen resulting in significant yield losses of animportant staple crop The ability of F virguliforme to function inthese two distinct roles suggests the need to consider the ge-nomic potential and ability of plant pathogens to express a gra-dation of transcriptional programs which in turn imparts lifestyleplasticity on a broad range of hosts

METHODS

Genome Sequencing Assembly and Annotation forFusarium virguliforme

PacBio and Illumina sequencing were performed using high molecularweightDNAextracted from lyophilized (FreeZone25 Labconco)Fusariumvirguliforme Mont-1 mycelia grown for 4 weeks in potato dextrose broth(Millipore-Sigma catalog no P6685) The F virguliforme Mont-1 isolatehas been extensively used as a model for the advancement of our un-derstandingof soybean (Glycinemax)SDS includingservingasamodel forgenomics and transcriptomics (Srivastava et al 2014 Ngaki et al 2016Sahuet al 2017) for theanalysis of pathogeneffector biology (Changet al2016a Chang et al 2016b) DNA was extracted using a modified cetyltrimethylammonium bromide procedure with 1 polyvinylpyrrolidone(Lade et al 2014) A PacBio library was constructed at the University ofGeorgiaGenomicsandBioinformaticsCore andsizeselected for 15- to20-kb fragmentsusing theBluePippin system (SageScientific) The librarywassequencedonaSequelPlatform and thesingle smart cell yielded65Gbofread data

For error correction Illumina TruSeq Nano DNA libraries were preparedand sequenced on an Illumina MiSeq v3 for a lane of 23 300 nucleotidesand HiSeq 4000 for a lane of 2 3 150 nucleotides at Michigan StateUniversity Research Technology Support Facility PacBio reads wereassembled and error corrected using Canu (v18 Koren et al 2017) usingdefault parameters with several modification including minReadLength5

2000 GenomeSize 5 51Mb minOverlapLength 5 1000 A default maxerror rate of 024 was used for alignment during error correction and 0045foroverlapandassemblyThemax target coveragewassetat403 Ak-mersizeof16wasused foroverlappingduringerror correction andak-mer sizeof 22 was used for overlapping during assembly

The genome size estimate for assembly was extrapolated from theprevious F virguliformeMont-1 draft genome of 509Mb (Srivastava et al2014) Due to the presence of contaminating bacterial DNA in the initialassembly draft contigs were compared with the published F virguliformegenome assembly with LAST (v912 Kiełbasa et al 2011) and novelcontigs were validated for fungal origin by BLAST1 (v2230 Camachoet al 2009) against the nonredundant National Center for BiotechnologyInformation (NCBI) database The genome graph structure was visualizedin Bandage (httpsacademicoupcombioinformaticsarticle31203350196114 Wick et al 2015) to survey contiguity and ambiguities(Supplemental Figure 1) Assembled contigs were error-corrected usingPilon (v122 Walker et al 2014) and default settings using a total of 503coverage of Illumina paired-end 300 nt and 150 nt data for F virguliformePaired end reads were adaptor and quality trimmed using Trimmomatic (v033 Bolger et al 2014) and then were aligned to the draft contigs usingBowtie2 (v226 Langmead and Salzberg 2012) with default settings Pilonwas run five times sequentially until limited (ie lt100) corrections werefound The new genome assembly was compared to the previous genomeassembly by QUAST (v30 Gurevich et al 2013) and is referred to asF virguliforme genome v2

Transcript evidence for gene predictions was acquired from both theNCBI (Short Read Archive [SRA] SRR1382101) and germinating

macroconidia from the F virguliforme RNA-seq time course (see below)Readswereadaptor andquality trimmedusingTrimmomatic (v033Bolgeret al 2014) for all transcript evidence These reads were then analyzedusing Breaker MAKER and Augustus gene model prediction algorithmshoused within FunGAP (v10 Min et al 2017) as transcript evidence Theparameters for running FunGAP were set as ndashsister_proteome Fusar-iumndashaugustus_species fusarium_graminearum with transcript readsprovided asndashtrans_read_single The resulting annotation from FunGAPconsisted of 16050 genes Single-copy fungal orthologs from Bench-marking Universal Single-Copy Orthologs (v3 Simatildeo et al 2015) wereused to assess the completeness of the genome annotation

Functional annotation was completed using Trinotate (v311 Bryantet al 2017) Trinotate-annotated gene models with evidence from severaldatabases (NCBI nonredundant protein database Swissprot-Uniprotdatabase GO and InterpoScan) with BlastX finding single hit at anE-value thresholdof 1E25 (Altschul et al 1990)Weused this information topredict protein domains with HMMER (v31 Finn et al 2011) trans-membrane proteins with TMHMM (v20 Krogh et al 2001) rRNA withRNAmmer (v12 Lagesen et al 2007) secreted proteins with SignalP(v41 Petersen et al 2011) and GO with GOseq (Young et al 2010)Additionally EffectorP (v20 Sperschneider et al 2016) was used topredict fungal effectorswithin the secreted proteins and dbCAN (Yin et al2012) was used to identify F virguliforme CAZymes and secondarymetabolism genes

Comparative Genomics with F virguliforme

MCSCAN toolkit (v11 Wang et al 2012) was used to identify syntenicgenepairs between the second version (v2) and the first version (v1) of theF virguliforme genome Conserved gene blocks were discovered throughLAST alignment Plots of macro- and micro-synteny were created by theMCScan in python

To discover novel and retained genes the v2 F virguliforme genomewas compared to the v1 F virguliforme genome Coding sequences forgenemodelswere extracted from the v1 genomeby gffread (Trapnell et al2010) Next gene sequences were reciprocally compared by BLASTn(v2226 Altschul et al 1990) Genes with an e-value below 1E25 greaterthan 70 gene alignment and 95 gene identity were classified as re-tainedgenes If a genewas alignedwith 95 identitywith an e-valuebelow1E25 but less than 70 gene alignment it was denoted as a mis-assembled gene Genes that were annotated in v2 that did not have analignment to the v1 genome were considered novel

Plant and F virguliforme Assays

SoybeancvSloan (providedbyMartinChilversMichiganStateUniversity)and maize (Zea mays) hybrid E13022S (Epley Brothers Hybrids Inc pro-videdbyMartinChilvers)weresurfacesterilized for30s in70(vv) ethanolfor 30 s and 10 (vv) bleach for 20 min and then triple rinsed in steriledistilled water for 1 min After sterilization soybean seeds were placedinside a Petri dish containing two sheets of sterile 100-mmWhatman filterpapersoaked in5mLofsterilewaterSoybeanseedswere incubated for5din total darkness at 21degC to enable germination Maize seeds were in-cubated insterilewater for 24h indarknessandplacedbetween twosheetsof sterile 100 mm Whatman filter paper with 5 mL of sterile water insidea Petri dish Seeds were incubated for 5 d in total darkness at 21degC toensure germination

F virguliformeMont-1 was propagated on potato dextrose agar (Difco)for 7weeks Spores of asexualmacroconidiawere collected diluted to 105

macroconidiamL21 in sterilewater and sprayed onto germinatedmaize orsoybean seedlings with an elongated a radicle using a 3-oz travel spraybottle Twenty-fivesprayswereapplied to theseedlingsatanglesof0deg90deg180deg and 270deg to that ensure seedswere thoroughly inoculated Formock

346 The Plant Cell

maintenance and survival of a pathogen in the absence ofa compatible host and another that supports proliferation andspread of a pathogen resulting in significant yield losses of animportant staple crop The ability of F virguliforme to function inthese two distinct roles suggests the need to consider the ge-nomic potential and ability of plant pathogens to express a gra-dation of transcriptional programs which in turn imparts lifestyleplasticity on a broad range of hosts

METHODS

Genome Sequencing Assembly and Annotation forFusarium virguliforme

PacBio and Illumina sequencing were performed using high molecularweightDNAextracted from lyophilized (FreeZone25 Labconco)Fusariumvirguliforme Mont-1 mycelia grown for 4 weeks in potato dextrose broth(Millipore-Sigma catalog no P6685) The F virguliforme Mont-1 isolatehas been extensively used as a model for the advancement of our un-derstandingof soybean (Glycinemax)SDS includingservingasamodel forgenomics and transcriptomics (Srivastava et al 2014 Ngaki et al 2016Sahuet al 2017) for theanalysis of pathogeneffector biology (Changet al2016a Chang et al 2016b) DNA was extracted using a modified cetyltrimethylammonium bromide procedure with 1 polyvinylpyrrolidone(Lade et al 2014) A PacBio library was constructed at the University ofGeorgiaGenomicsandBioinformaticsCore andsizeselected for 15- to20-kb fragmentsusing theBluePippin system (SageScientific) The librarywassequencedonaSequelPlatform and thesingle smart cell yielded65Gbofread data

For error correction Illumina TruSeq Nano DNA libraries were preparedand sequenced on an Illumina MiSeq v3 for a lane of 23 300 nucleotidesand HiSeq 4000 for a lane of 2 3 150 nucleotides at Michigan StateUniversity Research Technology Support Facility PacBio reads wereassembled and error corrected using Canu (v18 Koren et al 2017) usingdefault parameters with several modification including minReadLength5

2000 GenomeSize 5 51Mb minOverlapLength 5 1000 A default maxerror rate of 024 was used for alignment during error correction and 0045foroverlapandassemblyThemax target coveragewassetat403 Ak-mersizeof16wasused foroverlappingduringerror correction andak-mer sizeof 22 was used for overlapping during assembly

The genome size estimate for assembly was extrapolated from theprevious F virguliformeMont-1 draft genome of 509Mb (Srivastava et al2014) Due to the presence of contaminating bacterial DNA in the initialassembly draft contigs were compared with the published F virguliformegenome assembly with LAST (v912 Kiełbasa et al 2011) and novelcontigs were validated for fungal origin by BLAST1 (v2230 Camachoet al 2009) against the nonredundant National Center for BiotechnologyInformation (NCBI) database The genome graph structure was visualizedin Bandage (httpsacademicoupcombioinformaticsarticle31203350196114 Wick et al 2015) to survey contiguity and ambiguities(Supplemental Figure 1) Assembled contigs were error-corrected usingPilon (v122 Walker et al 2014) and default settings using a total of 503coverage of Illumina paired-end 300 nt and 150 nt data for F virguliformePaired end reads were adaptor and quality trimmed using Trimmomatic (v033 Bolger et al 2014) and then were aligned to the draft contigs usingBowtie2 (v226 Langmead and Salzberg 2012) with default settings Pilonwas run five times sequentially until limited (ie lt100) corrections werefound The new genome assembly was compared to the previous genomeassembly by QUAST (v30 Gurevich et al 2013) and is referred to asF virguliforme genome v2

Transcript evidence for gene predictions was acquired from both theNCBI (Short Read Archive [SRA] SRR1382101) and germinating

macroconidia from the F virguliforme RNA-seq time course (see below)Readswereadaptor andquality trimmedusingTrimmomatic (v033Bolgeret al 2014) for all transcript evidence These reads were then analyzedusing Breaker MAKER and Augustus gene model prediction algorithmshoused within FunGAP (v10 Min et al 2017) as transcript evidence Theparameters for running FunGAP were set as ndashsister_proteome Fusar-iumndashaugustus_species fusarium_graminearum with transcript readsprovided asndashtrans_read_single The resulting annotation from FunGAPconsisted of 16050 genes Single-copy fungal orthologs from Bench-marking Universal Single-Copy Orthologs (v3 Simatildeo et al 2015) wereused to assess the completeness of the genome annotation

Functional annotation was completed using Trinotate (v311 Bryantet al 2017) Trinotate-annotated gene models with evidence from severaldatabases (NCBI nonredundant protein database Swissprot-Uniprotdatabase GO and InterpoScan) with BlastX finding single hit at anE-value thresholdof 1E25 (Altschul et al 1990)Weused this information topredict protein domains with HMMER (v31 Finn et al 2011) trans-membrane proteins with TMHMM (v20 Krogh et al 2001) rRNA withRNAmmer (v12 Lagesen et al 2007) secreted proteins with SignalP(v41 Petersen et al 2011) and GO with GOseq (Young et al 2010)Additionally EffectorP (v20 Sperschneider et al 2016) was used topredict fungal effectorswithin the secreted proteins and dbCAN (Yin et al2012) was used to identify F virguliforme CAZymes and secondarymetabolism genes

Comparative Genomics with F virguliforme

MCSCAN toolkit (v11 Wang et al 2012) was used to identify syntenicgenepairs between the second version (v2) and the first version (v1) of theF virguliforme genome Conserved gene blocks were discovered throughLAST alignment Plots of macro- and micro-synteny were created by theMCScan in python

To discover novel and retained genes the v2 F virguliforme genomewas compared to the v1 F virguliforme genome Coding sequences forgenemodelswere extracted from the v1 genomeby gffread (Trapnell et al2010) Next gene sequences were reciprocally compared by BLASTn(v2226 Altschul et al 1990) Genes with an e-value below 1E25 greaterthan 70 gene alignment and 95 gene identity were classified as re-tainedgenes If a genewas alignedwith 95 identitywith an e-valuebelow1E25 but less than 70 gene alignment it was denoted as a mis-assembled gene Genes that were annotated in v2 that did not have analignment to the v1 genome were considered novel

Plant and F virguliforme Assays

SoybeancvSloan (providedbyMartinChilversMichiganStateUniversity)and maize (Zea mays) hybrid E13022S (Epley Brothers Hybrids Inc pro-videdbyMartinChilvers)weresurfacesterilized for30s in70(vv) ethanolfor 30 s and 10 (vv) bleach for 20 min and then triple rinsed in steriledistilled water for 1 min After sterilization soybean seeds were placedinside a Petri dish containing two sheets of sterile 100-mmWhatman filterpapersoaked in5mLofsterilewaterSoybeanseedswere incubated for5din total darkness at 21degC to enable germination Maize seeds were in-cubated insterilewater for 24h indarknessandplacedbetween twosheetsof sterile 100 mm Whatman filter paper with 5 mL of sterile water insidea Petri dish Seeds were incubated for 5 d in total darkness at 21degC toensure germination

F virguliformeMont-1 was propagated on potato dextrose agar (Difco)for 7weeks Spores of asexualmacroconidiawere collected diluted to 105

macroconidiamL21 in sterilewater and sprayed onto germinatedmaize orsoybean seedlings with an elongated a radicle using a 3-oz travel spraybottle Twenty-fivesprayswereapplied to theseedlingsatanglesof0deg90deg180deg and 270deg to that ensure seedswere thoroughly inoculated Formock

346 The Plant Cell

inoculated samples sterile distilled water was sprayed onto the seedlingsAfter inoculatedseedlingswere incubated for30min excess inoculumwasremoved and seedlings were incubated for an additional hour Followingincubation three maize or soybean seedlings were placed into sterilizedseed germination plastic pouches (CYG gemination pouch 165 cm3 18cmMega International) The pouchesweremoistenedwith 25mLof steriledistilled water Pouches containing seedlings were placed in a Bio-Chambers Bigfoot Series Model AC-60 growth chamber with 140 mEm22

s21 and1410 h lightdark cycle at 12degC for 7 d and then25degC for 7 d Plantswerewatered as neededwith sterile distilledwater Samples from tap rootsfrom soybean or radical roots from maize were taken at the same time ofday (1600 h) from the original 4-cm inoculation site throughout the timecourse The 2-week time course was repeated three independent times inthe same growth chamber with sampling of six pooled plants for RNAisolation and three plants for DNA isolation at 0 2 4 7 10 and 14 DAI ineach independent time Timepoint 0 (0DAI)was sampled immediately aftercompletion of fungal ormock inoculation andbefore transferring seedlingsto pouches Germinating macroconidia were sampled from an aliquot ofthe fungal inoculumusedabove for each independent experiment thatwascentrifuged at 20003 g to collect the germinated spores Six pooled rootsamples were generated for each independent run for each plant species(n5 6 for soybean or maize) and this was repeated for three independentruns (n5 18 for soybean andmaize) Additionally a sample of germinatingmacroconidia was collected for each independent run (n5 3) In total thisprovided 39 RNA samples (Supplemental Figure 8) Plant growth anddisease symptomologywas recorded at each time point by photographingwith a Nikon D50 camera

Fungal Colonization Analyses

To visualize fungal growth on samplesmicroscopic analyses ofmaize andsoybean roots were conducted at each time point for all treatments Rootswere cleared in 100ethanol followed by staining in a 005 (wv) trypanblue (Millipore Sigma catalog no T6146) solution containing equal parts ofwater glycerol and lactic acid (Savory et al 2012) Fungal structureswereobserved using an MZ16 dissecting scope (Leica)

RNA Extraction

Total RNA was isolated from 200 mg of ground flash-frozen germinatingmacroconidia and plant root samples and subsequently used for mRNA(mRNA) sequencing with an miRNeasy Mini Kit (Qiagen) Genomic DNAwas removed using the TURBODNase Free kit (Invitrogen) ExtractedRNAwasquantifiedusing theQubitRNABRkit (Invitrogen) andRNAqualitywasdeterminedbygel electrophoresis using the2100Bioanalyzer (Agilent)withthe Agilent RNA 6000 Pico kit

mRNA Library Preparation and Sequencing

Libraries were prepared using the Illumina TruSeq mRNA Library Prepa-ration Kit from three biological repeats of samples collected at each timepoint of F virguliformendash or mock-inoculated maize or soybean or germi-nating macroconidia samples by the Michigan State University ResearchTechnology Support Facility Pooled samples were sequenced on the Il-lumina HiSeq 4000 (single end 50 nucleotide mode) Base calling wasperformed using IlluminaReal TimeAnalysis (RTA) v277 and the output ofRTA was demultiplexed and converted to FastQ format with Illuminabcl2fastq v2191

Quantification of RNA-seq Expression and Differential Analysis

Reads were trimmed for adapter presence and quality score by Trimmo-matic (v033Bolger et al 2014) The trimmedreadswereuniquelymapped

to the corresponding reference genome of F virguliforme (Fv_v2) withHISAT2 (v 210 Kim et al 2015) using the following parametersndashdtandashrna-strandness F Hits from HISAT2 were converted from SAM to BAM formatby Picard (v 2181 httpbroadinstitutegithubiopicard) Alignments pergene model were counted by HTSeq (v061 Anders et al 2015 with thefollowing options ndashminaqual 50 -m intersection-strict -s reversendashidattr5-

gene_id Gene counts were imported into the R program DESeq2 (v1222Love et al 2014) normalized for library size and log2 transformed to de-termine correlation of biological replicates within each time point To assessbiological reproducibility we compared gene expression across biologicalreplicates andwe found gt90 reproducibility of the fungal gene expressionprofiles for the last time point of the infection time course indicating thebiological response of the fungus within each host was highly consistent(Supplemental Figure 2)

To determine differential gene expression patterns DESeq2 (v1222)with rawHTSeq countswas usedGeneswith less than 10 total rawcountsacross all samples were excluded To identify differentially expressedgenes (P 005) DESeq2 was used Through this approach two types ofpairwise comparison were performed (1) maize or soybean F virguliformein planta samples against germinating F virguliformemacroconidia acrosstime point at a log2 fold change gt2 to identify differentially induced genesand (2)F virguliforme gene expression patterns between hosts (iemaizeatDAI0versussoybeanat0DAI across timepoints) at a jlog2 foldchangegt1j to identify differentially induced genes

Analysis of Gene Coexpression Networks

Genes were filtered for weighted gene correlation network analysis(Langfelder andHorvath 2008) analysis (RDevelopmentCore Team 2010)for 90 of genes with less than 10 reads across all samples The resultant11112 genes were variance-stabilized transformed for importation andF virguliformendashsigned coexpression networks of maize or soybean wereconstructed separately A soft threshold power of 7 and tree cut height of015wereapplied tobothnetworksGeneexpressionwasclustered into22modules for F virguliforme colonization of maize and 20 modules forF virguliforme colonization of soybeanModuleswereplotted andvisualizedusing the R package ggplot2 (v311 Wickham 2016)

GO Enrichment Analysis

ToannotationGOterms foreachproteinannotated for theF virguliformev2genome unique GO terms from InterPro Scan (Jones et al 2014) wereextractedwith a custom script (httpsdoiorg105061dryad41ns1rn9q)Gene lists from either differential analysis or clusters from coexpressionanalysis were analyzed by TopGO (2340) conducted (Alexa andRahnenfuhrer 2018) in R Fisherrsquos Exact Test was conducted on eachgene set with a P 005 to determine significance of enrichment

Accession Numbers

The raw reads from the PacBio data Illumina DNA-seq and mRNA-seqreads can be downloaded at theNCBI SRA ThePacBio reads and IlluminaDNA-seq and RNA-seq reads are deposited to the NCBI SRA under Bi-oProjectPRJNA551448andPRJNA549951 respectivelyTheFvirguliformegenome assembly and annotation can be found at the Dryad DigitalRepository (httpsdoiorg105061dryad41ns1rn9q)

Supplemental Data

Supplemental Figure 1 Cartoon illustrating the genome assembly ofFusarium virguliforme

Supplemental Figure 2 Biological consistency of samples fromdifferent time courses

Fungal Transcriptional Plasticity Between Hosts 347

Supplemental Figure 3 Samples of fungal plant colonization groupby treatment

Supplemental Figure 4 Expression profiles of weighted gene coex-pression network modules from F virguliforme temporal colonizationof maize

Supplemental Figure 5 Expression profiles of weighted gene coex-pression network modules from F virguliforme temporal colonizationof soybean

Supplemental Figure 6 Temporal counts of Fusarium virguliformecandidate effector genes within soybean and maize hosts

Supplemental Figure 7 Temporal counts of Fusarium virguliformecarbohydrate active enzyme related genes within soybean andmaize hosts

Supplemental Figure 8 Overview of methodology to determinetranscriptomic profiles of Fusarium virguliforme during colonizationof maize or soybean

Supplemental Table 1 Genome assembly metrics of Fusariumvirguliforme Versions 1 and 2

Supplemental Table 2 GO enrichment of genes contained only in theFv_v2 genome

Supplemental Table 3 Number of quality trimmed reads uniquelyaligned to the Fusarium virguliforme genome v2

Supplemental Table 4 Host-specific differential gene expressionacross time points of Fusarium virguliforme colonization of soybean ormaize against germinating macroconidia

Supplemental Table 5 Host-specific upregulation of gene expressionwithin time points of Fusarium virguliforme colonization of soybean ormaize at log2 fold change gt 1

Supplemental Data Set 1 Gene ID conversion between v1 and v2 ofthe Fusarium virguliforme genome of conserved misassembledgenes and novel or missing genes in the genome

Supplemental Data Set 2 Gene list of candidate effectors generatedfrom EffectorP and SingalP

Supplemental Data Set 3 Gene list of carbohydrate active enzymesgenerated from CanDB

Supplemental Data Set 4 Gene Ontology enrichment of Fusariumvirguliforme gt 2 log2 fold change significant genes compared togerminating macroconidia on maize host

Supplemental Data Set 5 Gene Ontology enrichment of Fusariumvirguliforme gt 2 log2 fold change significant genes compared togerminating macroconidia on soybean host

Supplemental Data Set 6 Gene Ontology enrichment of Fusariumvirguliforme induced gene coexpression modules during maize rootcolonization

Supplemental Data Set 7 Gene Ontology enrichment of Fusariumvirguliforme induced gene coexpression modules during soybean rootcolonization

Supplemental Data Set 8 Differentially expressed genes at each timepoint of Fusarium virguliforme colonization between soybean andmaize with maize as a comparison base

Supplemental Data Set 9 Gene Ontology enrichment of Fusariumvirguliforme temporally differentially expressed genes within eithermaize or soybean root colonization

Supplemental Data Set 10 Effector list and Interpro scan annotation

Supplemental Data Set 11 CAZyme List and Interpro scanannotation

ACKNOWLEDGMENTS

We thank Kevin Childs and John Johnston for server access and compu-tational assistance andMartyChilvers forproviding theFusariumvirguliformeMont-1 isolate We would also like to recognize the support staff at theMichiganStateUniversity (MSU) Institute forCyber enabledResearchHighPerformance Computing Cluster for assistance in software optimizationThis research was supported by the MSU project GREEEN (GeneratingResearch and Extension to meet Economic and Environmental Needsgrant noGR16-008) by theCSMott Foundation (for fellowship support ofAB-Y) and by the MSU Plant Resilience Institute (grant no GR100125-Bean2)

AUTHOR CONTRIBUTIONS

AB-Y conducted experiments AB-Y and BD wrote the article ABYCMWRV-BBDdesigned the framework analyzeddata andprovidedcomments and editorial input during article preparation and revision

Received September 9 2019 revised November 11 2019 acceptedDecember 17 2019 published December 18 2019

REFERENCES

Alexa A and Rahnenfuhrer J (2018) topGO Enrichment analysisfor gene ontology Bioconductor Accessed Nov 12 2018

Altschul SF Gish W Miller W Myers EW and Lipman DJ(1990) Basic local alignment search tool J Mol Biol 215403ndash410

Anders S Pyl PT and Huber W (2015) HTSeq--a Pythonframework to work with high-throughput sequencing data Bio-informatics 31 166ndash169

Bae H Kim MS Sicher RC Bae H-J and Bailey BA (2006)Necrosis- and ethylene-inducing peptide from Fusarium oxysporuminduces a complex cascade of transcripts associated with signaltransduction and cell death in Arabidopsis Plant Physiol 1411056ndash1067

Bagnaresi P Biselli C Orrugrave L Urso S Crispino LAbbruscato P Piffanelli P Lupotto E Cattivelli L andValegrave G (2012) Comparative transcriptome profiling of the earlyresponse to Magnaporthe oryzae in durable resistant vs susceptiblerice (Oryza sativa L) genotypes PLoS One 7 e51609

Bolger AM Lohse M and Usadel B (2014) Trimmomatic Aflexible trimmer for Illumina sequence data Bioinformatics 302114ndash2120

Brown DW Butchko RA Busman M and Proctor RH (2007)The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisinproduction Eukaryot Cell 6 1210ndash1218

Brown DW and Proctor RH (2016) Insights into natural productsbiosynthesis from analysis of 490 polyketide synthases from Fu-sarium Fungal Genet Biol 89 37ndash51

Brown NA Evans J Mead A and Hammond-Kosack KE(2017) A spatial temporal analysis of the Fusarium graminearumtranscriptome during symptomless and symptomatic wheat in-fection Mol Plant Pathol 18 1295ndash1312

Bryant DM et al (2017) A tissue-mapped axolotl de novo tran-scriptome enables identification of limb regeneration factors CellReports 18 762ndash776

Caffall KH and Mohnen D (2009) The structure function andbiosynthesis of plant cell wall pectic polysaccharides CarbohydrRes 344 1879ndash1900

348 The Plant Cell

Camacho C Coulouris G Avagyan V Ma N PapadopoulosJ Bealer K and Madden TL (2009) BLAST1 Architecture andapplications BMC Bioinformatics 10 421

Cantarel BL Korf I Robb SMC Parra G Ross E MooreB Holt C Saacutenchez Alvarado A and Yandell M (2008)MAKER An easy-to-use annotation pipeline designed for emergingmodel organism genomes Genome Res 18 188ndash196

Chang HX Domier LL Radwan O Yendrek CR HudsonME and Hartman GL (2016a) Identification of multiple phyto-toxins produced by Fusarium virguliforme including a phytotoxiceffector (FvNIS1) associated with sudden death syndrome foliarsymptoms Mol Plant Microbe Interact 29 96ndash108

Chang HX Yendrek CR Caetano-Anolles G and HartmanGL (2016b) Genomic characterization of plant cell wall degradingenzymes and in silico analysis of xylanases and polygalacturonasesof Fusarium virguliforme BMC Microbiol 16 147

Chen Y Le X Sun Y Li M Zhang H Tan X Zhang D LiuY and Zhang Z (2017) MoYcp4 is required for growth conidio-genesis and pathogenicity in Magnaporthe oryzae Mol PlantPathol 18 1001ndash1011

Chowdhury S Basu A and Kundu S (2017) Biotrophy-necrotrophy switch in pathogen evoke differential response in re-sistant and susceptible sesame involving multiple signaling path-ways at different phases Sci Rep 7 17251

Cordovez V Mommer L Moisan K Lucas-Barbosa D PierikR Mumm R Carrion VJ and Raaijmakers JM (2017) Plantphenotypic and transcriptional changes induced by volatiles fromthe fungal root pathogen Rhizoctonia solani Front Plant Sci 8 1262

Demers JE Gugino BK and Jimeacutenez-Gasco MM (2015)Highly diverse endophytic and soil content genus-species Fusariumoxysporum populations associated with field-grown tomato plantsAppl Environ Microbiol 81 81ndash90

Derbyshire M et al (2017) The complete genome sequence of thephytopathogenic fungus Sclerotinia sclerotiorum reveals insightsinto the genome architecture of broad host range pathogens Ge-nome Biol Evol 9 593ndash618

Derntl C Kluger B Bueschl C Schuhmacher R Mach RLand Mach-Aigner AR (2017) Transcription factor Xpp1 isa switch between primary and secondary fungal metabolism ProcNatl Acad Sci USA 114 E560ndashE569

Elliott CE (2016) Control of gene expression in phytopathogenicAscomycetes during early invasion of plant tissue In Biochemistryand Molecular Biology The Mycota (A Comprehensive Treatise onFungi as Experimental Systems for Basic and Applied Research)Hoffmeister D ed Volume III (Cham Springer)

Fang YL Peng YL and Fan J (2017) The Nep1-like proteinfamily of Magnaporthe oryzae is dispensable for the infection of riceplants Sci Rep 7 4372

Finn RD Clements J and Eddy SR (2011) HMMER web serverInteractive sequence similarity searching Nucleic Acids Res 39W29-37

Giovannetti M Sbrana C Citernesi AS Avio L Gollotte AGianinazzi-Pearson V and Gianinazzi S (1994) Recognitionand infection process basis for host specificity of arbuscular my-corrhizal fungi In Impact of Arbuscular Mycorrhizas on SustainableAgriculture and Natural Ecosystems Gianinazzi S and SchuumleppH eds (Basel ALS Advances in Life Sciences Birkhaumluser)

Gupta A and Chattoo BB (2008) Functional analysis of a novelABC transporter ABC4 from Magnaporthe grisea FEMS MicrobiolLett 278 22ndash28

Gurevich A Saveliev V Vyahhi N and Tesler G (2013) QUASTQuality assessment tool for genome assemblies Bioinformatics 291072ndash1075

Hartman GL Chang HX and Leandro LF (2015) Researchadvances and management of soybean sudden death syndromeCrop Prot 73 60ndash66

Haueisen J Moumlller M Eschenbrenner CJ Grandaubert JSeybold H Adamiak H and Stukenbrock EH (2018) Highlyflexible infection programs in a specialized wheat pathogen EcolEvol 9 275ndash294

Heller J and Tudzynski P (2011) Reactive oxygen species inphytopathogenic fungi Signaling development and disease AnnuRev Phytopathol 49 369ndash390

Hoff KJ Lange S Lomsadze A Borodovsky M and StankeM (2016) BRAKER1 Unsupervised RNA-Seq-based genome an-notation with GeneMark-ET and AUGUSTUS Bioinformatics 32767ndash769

Horbach R Navarro-Quesada AR Knogge W and DeisingHB (2011) When and how to kill a plant cell Infection strategies ofplant pathogenic fungi J Plant Physiol 168 51ndash62

Jennings JC Apel-Birkhold PC Bailey BA and AndersonJD (2000) Induction of ethylene biosynthesis and necrosis in weedleaves by a Fusarium oxysporum protein Weed Sci 48 7ndash14

Jiang X Qiao F Long Y Cong H and Sun H (2017)MicroRNA-like RNAs in plant pathogenic fungus Fusarium oxy-sporum f sp niveum are involved in toxin gene expression finetuning 3 Biotech 7 354

Jones P et al (2014) InterProScan 5 Genome-scale protein func-tion classification Bioinformatics 30 1236ndash1240

Kiełbasa SM Wan R Sato K Horton P and Frith MC (2011)Adaptive seeds tame genomic sequence comparison Genome Res21 487ndash493

Kim D Langmead B and Salzberg SL (2015) HISAT A fastspliced aligner with low memory requirements Nat Methods 12357ndash360

Kleemann J Rincon-Rivera LJ Takahara H Neumann U VerLoren van Themaat E van der Does HC Hacquard SStuumlber K Will I Schmalenbach W Schmelzer E andOrsquoConnell RJ (2012) Sequential delivery of host-induced viru-lence effectors by appressoria and intracellular hyphae of thephytopathogen Colletotrichum higginsianum [published correctionappears in PLoS Pathog 2012 Aug8(8)] PLoS Pathog 8 e1002643

Kobayashi-Leonel R Mueller D Harbach C Tylka G andLeandro L (2017) Susceptibility of cover crop plants to Fusariumvirguliforme causal agent of soybean sudden death syndrome andHeterodera glycines the soybean cyst nematode J Soil WaterConserv 72 575ndash583

Koenning SR and Wrather JA (2010) Suppression of soybean yieldpotential in the continental United States by plant diseases from 2006 to2009 Plant Health Prog 11 101094php-2010ndash1122ndash1001-rs

Kolander TM Bienapfl JC Kurle JE and Malvick DK (2012)Symptomatic and asymptomatic host range of Fusarium virguli-forme the causal agent of soybean sudden death syndrome PlantDis 96 1148ndash1153

Koren S Walenz BP Berlin K Miller JR Bergman NH andPhillippy AM (2017) Canu Scalable and accurate long-read as-sembly via adaptive k-mer weighting and repeat separation Ge-nome Res 27 722ndash736

Krogh A Larsson B von Heijne G and Sonnhammer EL(2001) Predicting transmembrane protein topology with a hiddenMarkov model Application to complete genomes J Mol Biol 305567ndash580

Lade BD Patil AS and Paikrao HM (2014) Efficient genomicDNA extraction protocol from medicinal rich Passiflora foetidacontaining high level of polysaccharide and polyphenol Spring-erplus 3 457

Fungal Transcriptional Plasticity Between Hosts 349

Lagesen K Hallin P Roslashdland EA Staerfeldt H-H Rognes Tand Ussery DW (2007) RNAmmer Consistent and rapid anno-tation of ribosomal RNA genes Nucleic Acids Res 35 3100ndash3108

Lahrmann U Ding Y Banhara A Rath M Hajirezaei MRDoumlhlemann S von Wireacuten N Parniske M and Zuccaro A(2013) Host-related metabolic cues affect colonization strategies ofa root endophyte Proc Natl Acad Sci USA 110 13965ndash13970

Laluk K and Mengiste T (2010) Necrotroph attacks on plantswanton destruction or covert extortion Arabidopsis Book 8 e0136

Langmead B and Salzberg SL (2012) Fast gapped-read align-ment with Bowtie 2 Nat Methods 9 357ndash359

Lee Marzano S-Y Neupane A and Domier L (2018) Tran-scriptional and small RNA responses of the white mold fungusSclerotinia sclerotiorum to infection by a virulence-attenuating hy-povirus Viruses 10 713

Lofgren LA LeBlanc NR Certano AK Nachtigall J LaBineKM Riddle J Broz K Dong Y Bethan B Kafer CW andKistler HC (2018) Fusarium graminearum Pathogen or endo-phyte of North American grasses New Phytol 217 1203ndash1212

Lorrain C Marchal C Hacquard S Delaruelle C PeacutetrowskiJ Petre B Hecker A Frey P and Duplessis S (2018) Therust fungus Melampsora larici-populina expresses a conservedgenetic program and distinct sets of secreted protein genes duringinfection of its two host plants larch and poplar Mol Plant MicrobeInteract 31 695ndash706

Love MI Huber W and Anders S (2014) Moderated estimationof fold change and dispersion for RNA-seq data with DESeq2Genome Biol 15 550

Lysoslashe E Bone KR and Klemsdal SS (2008) Identification ofup-regulated genes during zearalenone biosynthesis in FusariumEur J Plant Pathol 122 505ndash516

Ma LJ et al (2010) Comparative genomics reveals mobile patho-genicity chromosomes in Fusarium Nature 464 367ndash373

Malcolm GM Kuldau GA Gugino BK and Jimeacutenez-GascoMdel M (2013) Hidden host plant associations of soilborne fungalpathogens an ecological perspective Phytopathology 103538ndash544

Min B Grigoriev IV and Choi IG (2017) FunGAP Fungal Ge-nome Annotation Pipeline using evidence-based gene model eval-uation Bioinformatics 33 2936ndash2937

Navi SS and Yang XB (2008) Foliar symptom expression in associ-ation with early infection and xylem colonization by Fusarium virguliforme(formerly F solani f sp Glycines) the causal agent of soybean suddendeath syndromePlant Heal Prog 101094PHP-2008ndash0222ndash01-RS

Ngaki MN Wang B Sahu BB Srivastava SK Farooqi MSKambakam S Swaminathan S and Bhattacharyya MK(2016) Transcriptomic study of the soybean-Fusarium virguliformeinteraction revealed a novel ankyrin-repeat containing defensegene expression of whose during infection led to enhanced re-sistance to the fungal pathogen in transgenic soybean plants PLoSOne 11 e0163106

OrsquoConnell RJ et al (2012) Lifestyle transitions in plant pathogenicColletotrichum fungi deciphered by genome and transcriptomeanalyses Nat Genet 44 1060ndash1065

Oliver RP and Ipcho SV (2004) Arabidopsis pathology breathesnew life into the necrotrophs-vs-biotrophs classification of fungalpathogens Mol Plant Pathol 5 347ndash352

Petersen TN Brunak S von Heijne G and Nielsen H (2011)SignalP 40 Discriminating signal peptides from transmembraneregions Nat Methods 8 785ndash786

R Development Core Team (2010) R A language and environmentfor statistical computing (Vienna Austria R Foundation for Statis-tical Computing)

Rai M and Agarkar G (2016) Plant-fungal interactions What triggersthe fungi to switch among lifestyles Crit Rev Microbiol 42 428ndash438

Sahu BB Baumbach JL Singh P Srivastava SK Yi X andBhattacharyya MK (2017) Investigation of the Fusarium virguli-forme transcriptomes induced during infection of soybean rootssuggests that enzymes with hydrolytic activities could play a majorrole in root necrosis PLoS One 12 e0169963

Sang H Chang H-X and Chilvers MI (2019) A Sclerotiniasclerotiorum transcription factor involved in sclerotial developmentand virulence on pea MSphere 4 e00615ndashe00618

Savory EA Adhikari BN Hamilton JP Vaillancourt B BuellCR and Day B (2012) mRNA-Seq analysis of the Pseudoper-onospora cubensis transcriptome during cucumber (Cucumis sat-ivus L) infection PLoS One 7 e35796

Schrettl M Bignell E Kragl C Sabiha Y Loss O EisendleM Wallner A Arst HN Jr Haynes K and Haas H (2007)Distinct roles for intra- and extracellular siderophores during As-pergillus fumigatus infection PLoS Pathog 3 1195ndash1207

Seidl MF Faino L Shi-Kunne X van den Berg GCM BoltonMD and Thomma BPHJ (2015) The genome of the sapro-phytic fungus Verticillium tricorpus reveals a complex effectorrepertoire resembling that of its pathogenic relatives Mol PlantMicrobe Interact 28 362ndash373

Selosse M-A Schneider-Maunoury L and Martos F (2018)Time to re-think fungal ecology Fungal ecological niches are oftenprejudged New Phytol 217 968ndash972

Shaw MW Emmanuel CJ Emilda D Terhem RB Shafia ATsamaidi D Emblow M and van Kan JA (2016) Analysis ofcryptic systemic Botrytis infections in symptomless hosts FrontPlant Sci 7 625

Simatildeo FA Waterhouse RM Ioannidis P Kriventseva EVand Zdobnov EM (2015) BUSCO Assessing genome assemblyand annotation completeness with single-copy orthologs Bio-informatics 31 3210ndash3212

Soanes DM Chakrabarti A Paszkiewicz KH Dawe AL andTalbot NJ (2012) Genome-wide transcriptional profiling of ap-pressorium development by the rice blast fungus Magnaporthe or-yzae PLoS Pathog 8 e1002514

Sperschneider J Gardiner DM Dodds PN Tini F CovarelliL Singh KB Manners JM and Taylor JM (2016) EffectorPPredicting fungal effector proteins from secretomes using machinelearning New Phytol 210 743ndash761

Srivastava SK Huang X Brar HK Fakhoury AM BluhmBH and Bhattacharyya MK (2014) The genome sequence ofthe fungal pathogen Fusarium virguliforme that causes suddendeath syndrome in soybean PLoS One 9 e81832

Stanke M Keller O Gunduz I Hayes A Waack S andMorgenstern B (2006) AUGUSTUS Ab initio prediction of alter-native transcripts Nucleic Acids Res 34 W435-9

Takahashi HK Toledo MS Suzuki E Tagliari L and StrausAH (2009) Current relevance of fungal and trypanosomatid gly-colipids and sphingolipids Studies defining structures conspicu-ously absent in mammals An Acad Bras Cienc 81 477ndash488

Trapnell C Williams BA Pertea G Mortazavi A Kwan Gvan Baren MJ Salzberg SL Wold BJ and Pachter L(2010) Transcript assembly and quantification by RNA-Seq revealsunannotated transcripts and isoform switching during cell differ-entiation Nat Biotechnol 28 511ndash515

Veloso J and van Kan JAL (2018) Many shades of grey in Bo-trytis host plant interactions Trends Plant Sci 23 613ndash622

Vollmeister E Schipper K Baumann S Haag C Pohlmann TStock J and Feldbruumlgge M (2012) Fungal development of theplant pathogen Ustilago maydis FEMS Microbiol Rev 36 59ndash77

350 The Plant Cell

Walker BJ Abeel T Shea T Priest M Abouelliel ASakthikumar S Cuomo CA Zeng Q Wortman J YoungSK and Earl AM (2014) Pilon An integrated tool for compre-hensive microbial variant detection and genome assembly im-provement PLoS One 9 e112963

Wang Y Tang H Debarry JD Tan X Li J Wang X LeeTH Jin H Marler B Guo H Kissinger JC and PatersonAH (2012) MCScanX A toolkit for detection and evolutionaryanalysis of gene synteny and collinearity Nucleic Acids Res 40 e49

Waterhouse RM Seppey M Simatildeo FA Manni M IoannidisP Klioutchnikov G Kriventseva EV and Zdobnov EM(2018) BUSCO applications from quality assessments to geneprediction and phylogenomics Mol Biol Evol 35 543ndash548

Wick RR Schultz MB Zobel J and Holt KE (2015) BandageInteractive visualization of de novo genome assemblies Bio-informatics 31 3350ndash3352

Wickham H (2016) ggplot2 Elegant graphics for data analysis(Springer-Verlag New York) Accessed April 11 2019

Yang F Li W and Joslashrgensen HJL (2013) Transcriptional re-programming of wheat and the hemibiotrophic pathogen Septoriatritici during two phases of the compatible interaction PLoS One 8e81606

Yang L Luumlbeck M and Luumlbeck PS (2015a) Effects of heterol-ogous expression of phosphoenolpyruvate carboxykinase andphosphoenolpyruvate carboxylase on organic acid productionin Aspergillus carbonarius J Ind Microbiol Biotechnol 421533ndash1545

Yang L Xie L Xue B Goodwin PH Quan X Zheng C LiuT Lei Z Yang X Chao Y and Wu C (2015b) Comparativetranscriptome profiling of the early infection of wheat roots byGaeumannomyces graminis var tritici PLoS One 10 e0120691

Yin Y Mao X Yang J Chen X Mao F and Xu Y (2012)dbCAN A web resource for automated carbohydrate-active enzymeannotation Nucleic Acids Res 40 W445-51

Young MD Wakefield MJ Smyth GK and Oshlack A (2010)Gene ontology analysis for RNA-seq Accounting for selection biasGenome Biol 11 R14

Zhang Y Tian L Yan D-H and He W (2018) Genome-widetranscriptome analysis reveals the comprehensive response of twosusceptible poplar sections to Marssonina brunnea infection Genes(Basel) 9 154

Zhao Z Liu H Wang C and Xu J-R (2013) Comparativeanalysis of fungal genomes reveals different plant cell wall de-grading capacity in fungi BMC Genomics 14 274

Fungal Transcriptional Plasticity Between Hosts 351

DOI 101105tpc1900697 originally published online December 18 2019 202032336-351Plant Cell

Amy Baetsen-Young Ching Man Wai Robert VanBuren and Brad DaySoybean

Transcriptional Plasticity Is Revealed by Host Colonization of Maize versuseFusarium virguliform

This information is current as of June 21 2020

Supplemental Data contentsuppl20191218tpc1900697DC1html

References content322336fullhtmlref-list-1

This article cites 84 articles 8 of which can be accessed free at

Permissions httpswwwcopyrightcomcccopenurldosid=pd_hw1532298Xampissn=1532298XampWTmc_id=pd_hw1532298X

eTOCs httpwwwplantcellorgcgialertsctmain

Sign up for eTOCs at

CiteTrack Alerts httpwwwplantcellorgcgialertsctmain

Sign up for CiteTrack Alerts at

Subscription Information httpwwwaspborgpublicationssubscriptionscfm

is available atPlant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists

DOI 101105tpc1900697 originally published online December 18 2019 202032336-351Plant Cell

Amy Baetsen-Young Ching Man Wai Robert VanBuren and Brad DaySoybean

Transcriptional Plasticity Is Revealed by Host Colonization of Maize versuseFusarium virguliform

This information is current as of June 21 2020

Supplemental Data contentsuppl20191218tpc1900697DC1html

References content322336fullhtmlref-list-1

This article cites 84 articles 8 of which can be accessed free at

Permissions httpswwwcopyrightcomcccopenurldosid=pd_hw1532298Xampissn=1532298XampWTmc_id=pd_hw1532298X

eTOCs httpwwwplantcellorgcgialertsctmain

Sign up for eTOCs at

CiteTrack Alerts httpwwwplantcellorgcgialertsctmain

Sign up for CiteTrack Alerts at

Subscription Information httpwwwaspborgpublicationssubscriptionscfm

is available atPlant Physiology and The Plant CellSubscription Information for

ADVANCING THE SCIENCE OF PLANT BIOLOGY copy American Society of Plant Biologists