j ourna l ho me pa ge: www.elsev ier .com/ locate /p lantsc i
omparative expression analysis of resistant and susceptible Populuslones inoculated with Septoria musiva
aiying Lianga,∗, Margaret Statonb, Yi Xua, Tao Xua, Jared LeBoldusc
Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634, USAClemson University Genomics Institute, Clemson, SC 29634, USADepartment of Plant Pathology, North Dakota State University, Fargo, ND 58108-6050, USA
r t i c l e i n f o
rticle history:eceived 28 September 2013eceived in revised form 24 February 2014ccepted 1 March 2014vailable online 16 March 2014
Septoria musiva is a major pathogen of Populus and can cause leaf spots and stem cankers in susceptibleclones. In order to investigate defense mechanisms of Populus in response to S. musiva, differential geneexpression in leaf tissues of two resistant (DN34, P. deltoides × nigra; NM6, P. nigra × maximowiczii) andtwo susceptible clones (DN164, P. deltoides × nigra; NC11505, P. maximowiczii × trichocarpa) was ana-lyzed by RNA-Seq. Of the 511 million reads obtained, 78% and 0.01% were successfully aligned to thegenomes of P. trichocarpa and S. musiva, respectively. Functional annotation of differentially expressedgenes based on comparisons between resistant and susceptible clones revealed that there were significantdifferences in the expression of genes involved in disease/stress resistance and oxidation–reduction inmock-inoculated leaves. Four days post inoculation with S. musiva, 36 differentially expressed genes werefound to be regulated in the same direction in both resistant clones. The 22 up-regulated loci in resistantclones included genes involved in protein fate, cell wall structure, and responsiveness to various biotic andabiotic stresses. In particular, Potri.008G187100 locus encodes a putative multi antimicrobial extrusionprotein and Potri.006G272600 encodes a family1 glycosyltransferase required for pathogen resistance.The differentially expressed loci with increased expression in the susceptible clones corresponded toNB-ARC domain-containing disease resistance protein, phospholipase A 2A, MutT/nudix family protein,and an elicitor-activated gene 3-1 product. The results from this study indicate that strong defense
mechanisms involved in oxidation–reduction, protein fate, secondary metabolism, and accumulation ofdefense-related gene products may contribute to Septoria resistance in DN34 and NM6, while increasedexpression of hypersensitive response-loci, particularly those encoding NB-ARC domain-containing dis-ease resistance proteins, may contribute to the susceptibility of DN164 and NC11505 through interactionwith pathogen effectors.
Populus comprises about thirty economically and ecologicallymportant species worldwide [1], including black poplars, cotton-
oods, and aspens. Populus species and their hybrids are amonghe fastest-growing temperate trees. The primary commercial usesf poplar trees include pulpwood, engineered lumber products,
nd bioenergy [2]. In anticipation of shrinking petroleum reservesnd a reduced land base to produce forest products, interestn short-rotation intensive poplar culture as an alternative fuel
∗ Corresponding author at: Poole Agricultural Center, Room 153, 130 McGintyourt, Department of Genetics and Biochemistry, Clemson University, Clemson, SC9634, USA. Tel.: +1 864 656 2414.
and fiber resource has increased. For instance, plantations havebeen established in Oregon, Washington, the north central UnitedStates, the southern Mississippi river valley, and three Canadianprovinces (Alberta, Quebec, and British Columbia) and are impor-tant sources of fiber in those regions [3–6]. However, widespreadadoption of short-rotation intensive poplar production is hin-dered by the occurrence of several important foliar and steminfecting pathogens [7]. Among them, leaf spot and stem cankercaused by the necrotrophic fungus Septoria musiva Peck (Teleo-morph = Mycosphaerella populorum G.E. Thompson) [Sphaerulinamusiva (Peck) Quaedivlieg, Verkley & Crous: proposed new genus]([8] and references therein) is the most serious disease affecting
hybrid poplar production in North America (review in [7,9]). Severeleaf spot outbreaks can reduce the photosynthetic area and causepremature defoliation, thereby decreasing annual growth. Stemcankers may reduce growth, predispose the tree to colonization
y secondary organisms, and cause severe girdling, increasing theisk of wind breakage of the main stem. Biomass losses due to thisathogen vary among clones and have been reported to be as highs 63% of total yield [10]. Furthermore, Ostry et al. [11] found that6% of the clones in a plot located near infected plantations inichigan had cankers five years after plot establishment. A field
rial conducted in Canada found that all 56 genotypes of P. bal-amifera had at least one stem canker per stem seven years afterlanting [12]. Similar impacts have been observed in other studies13–16].
Chemical and biological control of Septoria leaf spot and stemanker has been attempted. For example, benomyl applied monthlythree applications per growing season) or on a bimonthly sched-le (five applications per growing season) beginning at leaf flusheduced the incidence of Septoria canker [14]. Three applicationsf benomyl during the growing season reduced foliar leaf diseaseaused by S. musiva [17]. Yang et al. [18] reported that pre- andost-treatment of P. berolinensis with Phaeotheca dimorphospora, aeuteromycotina fungus, could reduce the severity of leaf spot in
he greenhouse. Spraying disease-suppressive Streptomyces strainsan also significantly reduce leaf disease caused by S. musiva [19].owever, these approaches are expensive due to the application
requency required for efficient control. In the case of Streptomyces,pore application needed to be done at least monthly but prefer-bly weekly during periods of S. musiva spore release. As a result,lanting resistant clones appears to be the best means of managingeptoria leaf spot and stem canker.
A successful disease-resistance breeding program relies on annderstanding of the genetic basis of resistance and interactionsetween the host and the pathogen. However, genetic informationn Septoria resistance in Populus is scarce, despite the economicnd ecological importance of the diseases. It is known that east-rn cottonwood (Populus deltoides Batr. ex Marsh) is resistant toanker, while P. trichocarpa Torr. & Gray, indigenous to the westoast of North America, and the Asian species P. maximowicziienry, are generally susceptible to Septoria canker [13,20]. Hybridrosses of P. deltoides with poplars in the section Tacamahaca arearticularly susceptible to canker [13,20]. Newcombe and Ostry20] confirmed the hypothesis that the resistance to S. musivaanker inherited from P. deltoides in F1 and F2 progeny obtainedrom crosses with P. trichocarpa was recessive. The authors alsoonfirmed the absence of a link between inherited factors in P.eltoides conferring resistance to S. musiva canker and those confer-ing quantitative resistance to leaf spot disease caused by Septoriaopulicola Peck, a closely related pathogen. Evidence for three quan-itative trait loci of major effect in resistance to S. populicola wasbtained by Newcombe & Bradshaw [21]. They revealed that theesistance alleles were contributed by the P. deltoides parent, andere dominant to the P. trichocarpa alleles. These are the only two
eports on the inheritance of Septoria resistance in hybrid poplar.o such genetic information is available for leaf spot caused by S.usiva.
The recent development of high-throughput sequencing tech-ologies provides an unprecedented opportunity to explore theefense response thoroughly by whole-genome expression pro-le analysis. In this study, we conducted the first global analysisf the Populus defense transcriptome dynamics in response toeaf spot caused by S. musiva using the RNA Sequencing method-logy. RNA sequencing, or RNA-Seq, is increasingly being usedor global gene expression profiling because it allows unbiaseduantification of expression levels of transcripts with a higherensitivity and broader genome coverage than microarrays [22].
n plants, this technology has been used to identify pathogenesponse genes in a few species, including cotton [23], soybean [24],rabidopsis [25], and Populus (infected with rust pathogen Melamp-ora larici-populina) [26]. We isolated RNA from inoculated and
e 223 (2014) 69–78
mock-inoculated leaf tissues from two resistant and two sus-ceptible hybrid poplar clones. We constructed a total of 16cDNA libraries. Differential expression analyses were performedbetween resistant and susceptible clones and inoculated and mock-inoculated leaf tissues. Our study was the first attempt to identifyand understand poplar gene expression in response to leaf inoc-ulation by S. musiva. The overall goal was to elucidate defensemechanisms in Populus in response to S. musiva. Such informa-tion is crucial in order to understand compatible and incompatiblehost–pathogen interactions and inform strategies to optimize solu-tions and minimize losses to S. musiva and other necrotrophicpathogens.
Materials and methods
Host propagation
Dormant branch cuttings from 14 clones of hybrid poplar(Table 1) were collected in February 2012, from the previous year’sgrowth, at the University of Wisconsin–Madison research farm. Thebranches were cut into 10-cm-long sections, soaked in de-ionizedwater for 48 h at 4 ◦C, and planted into 21-cm-deep SC10 supercone-tainersTM (Stuewe & Sons® Deepots D40 cell; Stuewe & SonsInc., Tangent, OR) with only the top-most bud exposed above thegrowing medium surface (SunGro® Professional Mix #8; SunGroHorticulture® Ltd., Agawam, MA). The SunGro® mix was amendedwith Nutricote® 200 day slow release 15-9-12 fertilizer (6.5% NO3-N, 6.5% NH4-N, 13% P2O5, 13% K2O, 1.2% Mg, 0.02% B, 0.05% Cu,0.2% chelated Fe, 0.06% Mn, 0.02% Mo, 1.3% EDTA; Plant ProductsCompany Ltd., Brampton, ON) and supplemented with a 250 ppmsolution of 20-20-20 fertilizer on a weekly basis (Plant ProductsCompany Ltd., Brampton, ON). The cuttings were then placed intoa greenhouse maintained at 20/15 ◦C (day/night) with an 18 h pho-toperiod supplemented with artificial lights. Following the initial90 days of growth trees were transplanted from the cone-tainersinto 1 gallon plastic pots (Listo Products Ltd., Vancouver, BC) con-taining SunGro® mix amended with Nutricote® and fertilized on aweekly basis.
Pathogen propagation
The three isolates of S. musiva used in this experiment were iso-lated from Septoria cankers collected from dormant P. delotides × P.nigra hybrids at Verso Paper Company’s Belle River Nursery nearAlexandria Minnesota (approximate location: Lat: 45.99; Long:95.23). Isolations were made by surface disinfesting the bark in a5% bleach solution and then rinsing twice in sterile distilled waterfor 2 min. Subsequently, bark was removed from the canker mar-gin, and a sliver of necrotic tissue was placed on a Petri dish withK-V8 medium (180 ml V8 juice, Campbell Soup Company, Camden,NJ, USA; 2 g calcium carbonate; 20 g agar, Difco TM, Franklin Lakes,NJ, USA; and 820 ml de-ionized water amended with 300 mg l−1
A randomized complete block design with 10 blocks was uti-lized (8 “Septoria inoculated” + 2 “mock inoculated”). Each hybridpoplar clone occurred once per block. For each isolate inocu-lum was produced by dislodging conidia in sterile distilled water
H. Liang et al. / Plant Science 223 (2014) 69–78 71
Table 1Resistance and susceptibility to S. musiva in poplar clonesa.
Clone Genotypeb Mean tree height (cm) Mean # of lesions Canker severityc (# lesions/cm) Leaf spot severityd
a The values were based on an average across 5 inoculated blocks. The mean disease severity of each clone measured was the number of lesions (cankers) per cm of stemat the time of inocualtion.
b Numbers refer to Populus spp.: 1 = balsamifera, 4 = deltoides, 5 = deltoides var. angulata, 6 = maximowiczii, 7 = nigra, 9 = nigra var. caudina, 10 = nigra var. charkowiensis,1 ).
feebmeobttgsabtTUfawpSt
I
(fsrrwouirbwgcfGr
1 = nigra var. incrassata, 13 = trichocarpa, and 14 = tristis (Weiland and Stanosz 2007c Severity values with different letters are significantly different (p < 0.05).d L: low scale (few small spots); H: high scale (many large spots).
rom 14-day-old colonies on K-V8 medium. The concentration ofach isolate was corrected to 1 × 106 spores ml−1. Subsequently,qual volumes of each isolate were combined to create a finalulked spore suspension for inoculations. Initially, tree height waseasured and inoculations were conducted as described in the lit-
rature [28]. Briefly, inoculum was sprayed on the stem and leavesf each tree (∼50 ml tree−1) which were then placed in separatelack plastic bags with two pieces of moist paper towel. The plas-ic bags were sealed for 48 h at approximately 22 ◦C after whichime the trees were returned to their original positions in thereenhouse. The mock-inoculated trees were treated exactly in theame manner except sterile distilled water was used instead of
spore suspension. Twenty-one days after inoculation the num-er of necrotic stem lesions per tree was counted and divided byree height at the time of inoculation to determine disease severity.he MIXED procedure in SAS [29] was used to calculate Best Linearnbiased Estimates (BLUEs) for each clone across all 5 blocks used
or phenotyping. The model used for BLUEs estimation had clone as fixed effect, block as a random effect, and the repeated statementas used to model heterogeneous variances among clones. Multi-le comparisons among clones were conducted using Tukey’s Leastignificant Difference [29]. Leaf spot severity was scored on a Lowo High scale (Low = few small spots and High = many large spots).
llumina sequencing and data processing
Four days after inoculation, leaves were collected from 4 blocks3 “Septoria-inoculated” + 1 “mock-inoculated”) and immediatelyrozen in liquid nitrogen. Three of the blocks were randomlyelected from the 8 “Septoria-inoculated” blocks and 1 wasandomly selected from the 2 “mock-inoculated” blocks. Theemaining 6 blocks (5 “Septoria-inoculated” + 1 “mock-inoculated”)ere kept in the greenhouse for leaf spot and stem canker phen-
typing as described above. All samples were stored at −80 ◦Cntil used for RNA extraction. Total RNA was extracted accord-
ng to the protocol of Meisel et al. [30] with a modification ofeplacing spermidine trihydrochloride with 0.1% diethyl pyrocar-onate (DEPC). The integrity and quality of RNA were verifiedith an Agilent Technologies 2100 Bioanalyzer (Agilent Technolo-
ies) before subjecting the RNA to cDNA synthesis and library
onstruction. cDNA synthesis and library construction were per-ormed by the Georgia Genomics Facility at The University ofeorgia and followed the Illumina TruSeq® RNA Sample Prepa-
ation v2 Guide. Poly-T oligo attached magnetic beads were used
in two rounds of messenger RNA isolation and a centrifuge stepwas included to remove the majority of the ribosomal and othernon-messenger RNA as described in the protocol. Based on theleaf spot severity observed 21 days after inoculation, four hybridpoplar clones were selected for transcriptome analysis: two resis-tant (DN34, P. deltoides × nigra, and NM6, P. nigra × maximowiczii)and two susceptible (DN164, P. deltoides × nigra, and NC11505, P.maximowiczii × trichocarpa). There were three inoculated and onemock-inoculated trees per clone. A total of 16 cDNA samples wereindividually bar-coded and run in two lanes of HiSeq 2000 v3. Theraw reads are deposited in the Sequence Read Archive of NationalCenter for Biotechnology Information with a study accession num-ber of SRP033272 (Bioproject# PRJNA229492).
The P. trichocarpa genome and gff annotation file (version210) were downloaded from Phytozome (ftp://ftp.jgi-psf.org/pub/compgen/phytozome/v9.0/Ptrichocarpa/). The S. musiva genomeand gff annotation file (SO2202 v1.0) were downloaded from JGI(http://genome.jgi-psf.org/pages/dynamicOrganismDownload.jsf?organism=Sepmu1). The RNA-Seq reads were aligned to both ref-erence genomes using the software package TopHat (version2.0.8b) with parameters: mate inner distance of 1, minimumintron length of 35, and library type fr-unstranded [31]. Rawcounts for each gene were obtained by running HTSeq v0.5.3p9(http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html). Functional annotation of the previously annotated poplargenes was taken from the Phytozome site.
Identification of differentially expressed (DE) genes
A statistical package, DESeq (version 1.10.0) was used to esti-mate effective library size (method estimateSizes, default values),estimate the dispersion of the data (method estimateDispersions,default values) and to compare two conditions using a nega-tive binomial distribution (method nbinomTest, default values)[32]. Where conditions had biological replicates, those replicateswere taken into account. An adjusted p-value of less than 0.05was considered significant across all comparisons. Dispersion plotsfor the inoculated samples are listed in Supplemental Material 1.Gene Ontology (GO) terms were determined based on TAIR anno-
tation (http://www.arabidopsis.org/tools/bulk/go/index.jsp), andfunctional categorization of differentially expressed transcriptswas scored by annotation for putative molecular functions, cellu-lar components and biological processes. GO enrichment analysis
as performed using the DAVID Bioinformatics Resources 6.7http://david.abcc.ncifcrf.gov/).
ata validation by qRT-PCR
Thirteen differentially expressed genes identified by the RNA-eq approach were randomly selected for primer design withrimer3 [33]. The sequences of primers are listed in Supplementalaterial 2, with four pairs being able to amplify intron sequences,
erving as a genomic DNA contamination control. cDNAs wereynthesized using a high-capacity cDNA Reverse Transcription kitApplied Biosystems, CA) with the same total RNA isolated for RNA-eq analysis. Each quantitative RT-PCR reaction was carried out in
total volume of 20 �l, containing 10 �l of 2× IQ SYBR green super-ix (Bio-Rad, CA), 1 �l of cDNA template (1000 ng �l−1), and 1 �l of
0 �M of each gene-specific primer. The reactions were conductedn a Stratagene MX3000p QPCR system (Agilent, CA) with the cycleetting as follows: 95 ◦C denaturation for 10 min, 40 cycles of 95 ◦Cenaturation for 30 s, 55 ◦C annealing for 30 s and 72 ◦C extensionor 30 s. A Populus tubulin A (TUA) was used as the internal standards it had a lower stability index based on the analysis described inrunner et al. [34]. At least three technical replicates were included
or each sample. A melting curve step was included to check theinding specificities of the primers. Data were analyzed with Strata-ene MxPro QPCR software and a standard curve method was usedo calculate the Ct values [35]. The R squared was calculated basedn the Spearman correlation [36]. For the qPCR measurements, theatio of the average condition 1 values to average condition 2 valuesas used. For the sequencing measurements, the correlation was
ased on the ratio of condition 1 to condition 2 as calculated byESeq, which performed normalization and a variance-stabilizing
ransformation across all genes.
esults
isease severity of hybrid poplar clones inoculated with S. musiva
Disease severity of leaf spots and stem infections was eval-ated three weeks post inoculation. The most resistant hybridoplar genotypes were DN34 in terms of both stem and leaf infec-ions (0.11 lesions cm−1 on stems; low leaf spot severity (L)) andM6 (0.18 lesions cm−1; L) (Table 1). For both of these genotypes
here were few stem lesions and a low number of small leaf spots.he most susceptible genotypes were NC11505 (2.06 lesions cm−1;igh leaf spot severity (H)) and DN164 (0.79 lesions cm−1; H)Table 1), in which there were a large number of stem lesions andhere were many large leaf spots. In addition, we detected a signif-cant positive relationship between growth rate and susceptibilityR-squared = 0.34).
nalysis of RNA-Seq data
A total of 16 cDNA libraries were prepared from clones DN34,M6, NC11505, and DN164, representing 2 resistant and 2 sus-eptible genotypes. Twelve inoculated and 4 mock-inoculated leafamples were used. The cDNA libraries were subjected to 2 lanes ofNA-Seq PE100. In total, 511 million reads were generated, consti-uting 22.8 Gb of sequence (Table 2). An average of 78% of the readsas successfully aligned to the P. trichocarpa genome, 23% of whichere uniquely aligned. The low percentage of uniquely aligned
eads may reflect the relatively recent Salicoid whole genomeuplication that is shared across the Salicaceae [37]. All annotated
oci from poplar, 41,291, have at least one aligned read in this study.n addition, 1392 putative new areas of transcription were identi-ed. Among the 44.9 million reads (22% of the total reads) that didot map to the P. trichocarpa genome, 88.2% could be assembled Ta
nto contigs. When queried in Swiss-Prot, 68.7% and 1.9% of thessembled reads were mapped to a plant sequence and a microbialequence, respectively, while 26.5% did not have a match. Whenapped to the genome of S. musiva, 0.01% of the reads from the
noculated leaves were alignable, 62.2% of which were mappedo a gene and 70.8% mapped uniquely. Very few reads from the
ock-inoculated leaves, ranging from 795 to 1162, were alignableo the pathogen’s genome and none of these reads was mapped to
gene-coding region.
ifferentially expressed Populus genes betweeneptoria-inoculated leaves and mock-inoculated leaves within theame clone
There were five DE genes (all down-regulated) in S. musiva-hallenged leaves from clone DN34, seven (three in up-regulatednd four in down-regulated) from NM6, and seven (all down-egulated) from NC11505 (Table 3 and Supplemental Material 3)hen compared to the mock-inoculated leaves. Genes encoding
Matrixin family protein, a phosphorylase superfamily protein, basic chitinase, a Kunitz family trypsin and protease inhibitorrotein, and an alpha/beta-hydrolases superfamily protein wereifferentially expressed in DN34. Laccase 17, a cellulose synthaseene, and an arabinogalactan protein 18 gene were up-regulatedn NM6 Septoria-inoculated leaves, while genes encoding a glyco-yltransferase family 61 protein, a zinc transporter 1 precursor, arolyl oligopeptidase family protein, and an unknown protein wereown-regulated. In NC11505, a highly susceptible clone, in addi-ion to natural resistance-associated macrophage protein 3 gene,BP3-responsive gene 1, and 1-cysteine peroxiredoxin 1 gene, fournknown genes showed a decreased transcriptional level in inoc-lated leaves. For DN164 the recommended parametric dispersiont did not succeed.
When combining the data from both the resistant clones (DN34nd NM6) or susceptible clones (DN164 and NC11505), sevenenes were significantly down regulated in the resistant cloneshen compared to mock-inoculated leaves, while there was only
ne (Potri.005G096200, no annotation available) in the suscep-ible clones (Supplemental Material 3). One of the seven genesdentified as differentially expressed in the two resistant clonesad no significant match in any public database. The closest Ara-idopsis homologues of the remaining six genes were AT1G76140Prolyl oligopeptidase family protein), AT3G12500 (basic chiti-ase), AT1G17860 (Kunitz family trypsin and protease inhibitorrotein), AT3G12750 (zinc transporter 1 precursor), AT4G18550alpha/beta-hydrolases superfamily protein), and AT2G41640 (gly-osyltransferase family 61 protein).
ifferentially expressed Populus genes identified in comparisonsmong mock-inoculated leaves from different clones
In mock-inoculated leaves, DE genes were found in compar-sons between clones, with the DN164 vs NC11505 having the mostE genes (122) and NM6 vs DN164 the fewest (43) (Supplementalaterial 3). There were 31 common DE genes between the mock-
noculated leaves of the resistant clones (DN34 and NM6) and theighly susceptible NC11505. All 31 DE genes were up or downegulated in the same direction between DN34 and NM6 (Supple-ental Material 4). Eight of these DE genes were identified as part
f the oxidation–reduction biological process (GO:0055114). ThisO term had the highest enrichment score of 1.23 (fold enrichment:.7, p value: 0.0006). Two defense response genes (AT1G74100, sul-
otransferase 16 and AT1G70830, MLP-like protein 28) were downegulated in NC11505. Similarly, there were 11 common DE genesetween the comparisons of resistant clones (DN34 and NM6) vshe susceptible clone DN164 in the mock-inoculated leaves, and
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these DE genes had the same up or down regulation between DN34and NM6 (Supplemental Material 4).
Differentially expressed Populus genes identified in comparisonsof different inoculated clones
Compared to mock-inoculated leaves, there were more DE genesin leaves challenged with the pathogen, with the exception of com-parisons between the two susceptible clones, DN164 vs NC11505,in which no DE genes were identified (Supplemental Material 3).The number of DE genes in the other comparisons ranged from 550(NM6 vs DN164) to 1499 (DN34 vs NC11505), with all comparisonshaving cellular processes, enzyme activity, and cytoplasmic compo-nents as the major GO terms (Supplemental Material 5). Hydrolaseactivity, protein binding, DNA or RNA binding, cytoplasmic com-ponents, and unknown GO molecular function scored higher in theDE genes identified in comparisons with DN34 (DN34 vs NC11505,vs DN164, and vs NM6) than the ones resulting from comparisonswith NM6 (NM6 vs NC11505, and vs DN164), while the latter hada higher percentage in metabolic processes as well as in mem-brane and extracellular components (Supplemental Material 5).There were 37 common DE loci among comparisons of resistant vssusceptible clones, 36 of which were regulated in the same direc-tion in both resistant clones (Table 4). The 22 up-regulated DE lociincluded a gene participating in protein fate (trypsin family pro-tein gene), a gene involved in cell wall structure (FASCICLIN-likearabinogalactan 6), and 13 genes responsive to various biotic andabiotic stresses. In particular, Potri.008G187100 locus encodes aputative multi antimicrobial extrusion protein; Potri.006G272600encodes a family1 glycosyltransferase required for pathogen resis-tance, and Potri.012G100600 encodes an F-box/LRR-repeat proteinconferring disease resistance.
The 14 down-regulated loci were enriched with genes encod-ing serine/threonine-protein kinases (enrichment Score = 2.27, foldenrichment = 15.8, p value = 1.37E−04), including a leucine-richrepeat Resistance (R) protein encoding gene (Potri.017G035500).Five of the DE loci did not have an Arabidopsis match. In addi-tion, comparisons of different clones challenged with S. musivaidentified 12 loci involved in plant-type hypersensitive responses(GO:0009626), among which 4 NB-ARC domain-containing diseaseresistance protein-encoding loci were down-regulated and 2 wereup-regulated in the resistant clones (Supplemental Material 6).
Validation of RNA-Seq-based gene expression by qRT-PCR
To validate the RNA-Seq-based gene expression levels thatcorrelated with the number of reads obtained, qPCR was per-formed on 13 randomly selected genes with different expressionlevels and functional assignments, resulting in 77% having thesame up or down regulation as the RNA-Seq results (SupplementalMaterial 2). Overall, results from the qPCR and RNA-Seq analyseswere moderately consistent. The correlation of expression mea-surements of all 13 genes between RNA-Seq and qRT-PCR waslow (R-squared = 0.13). However, when only inoculated sampleswere examined, which contained 3 replicates the R-squared was0.97 indicating that sequence replicates are necessary for accurateexpression measurements.
S. musiva sequences
Isolation of transcripts from inoculated tissues did not gener-ate enough S. musiva reads for statistical differential expression
analysis. The common loci identified between two host clonesranged from 59 to 75. A total of 31 loci were found in all fourclones, 18 of which were mapped to a gene. These includedgenes responsive to oxidative stress (oxidoreductase activity and
74 H. Liang et al. / Plant Science 223 (2014) 69–78
Table 3Differentially expressed Populus genes identified in comparison of S. musiva-inoculated and mock-inoculated leaves.
P. trichocarpa ID Best Arabidopsis match Expression comparison(un-infected vs infected)
Adjustedp-value
DN34 013G100400 AT1G59970.1: matrixin family protein 6.66 0.00019G050200 AT4G24340.1: phosphorylase superfamily protein 7.01 0.02009G141800 AT3G12500.1: basic chitinase 15.94 0.00010G007900 AT1G17860.1: Kunitz family trypsin and protease inhibitor protein 35.63 0.01015G026500 AT4G18550.1: alpha/beta-hydrolases superfamily protein 89.80 0.00
NM6 011G120300 AT5G60020.1: laccase 17 0.00 0.00018G103900 AT5G17420.1: cellulose synthase family protein 0.04 0.05009G092300 AT4G37450.1: arabinogalactan protein 18 0.05 0.05016G132100 11.34 0.05016G057000 AT2G41640.1: glycosyltransferase family 61 protein 24.61 0.05010G173300 AT3G12750.1: zinc transporter 1 precursor 25.06 0.00002G014000 AT1G76140.1: prolyl oligopeptidase family protein 32.15 0.00
Table 4Common differentially expressed Populus genes among comparisons of resistant clones vs susceptible clones in S. musiva-inoculated leaves.
P. trichocarpa ID Direction of regulation inresistant clonea
Top Arabidopsis match Annotation
012G100600 Up AT5G63520.1 F-box/LRR-repeat protein015G054800 Up NA018G001500 Up AT5G27660.1 Trypsin family protein with PDZ domain011G021200 Up NA013G120600 Up AT2G20520.1 FASCICLIN-like arabinogalactan 6011G032300 Up AT1G61680.1 Terpene synthase 14014G094600 Up AT2G16070.2 Plastid division2003G082100 Up AT5G02890.1 HXXXD-type acyl-transferase family protein006G272600 Up AT2G15480.1 UDP-glucosyl transferase 73B5001G191000 Up AT4G25150.1 HAD superfamily, subfamily IIIB acid phosphatase005G237600 Up AT5G06300.1 Putative lysine decarboxylase family protein010G137100 Up AT1G71691.1 GDSL-like lipase/acylhydrolase superfamily protein002G189300 Up AT2G34430.1 Light-harvesting chlorophyll–protein complex II subunit B1,
senescence-repressed002G154900 Up NA008G061800 Up AT5G05960.1 Bifunctional inhibitor/lipid-transfer protein/seed storage 2S albumin
superfamily protein017G098000 Up NA018G072000 Up AT3G24450.1 Heavy metal transport/detoxification superfamily protein008G187100 Up AT3G26590.1 MATE efflux family protein001G272700 Up AT5G62690.1 Tubulin beta chain 2T063200 Up AT4G15450.1 Senescence/dehydration-associated protein-related, similar to
early-responsive to dehydration stress ERD7018G111900 Up AT4G19810.1 Glycosyl hydrolase family protein with chitinase insertion domain001G024900 Up AT2G38750.1 Annexin 4017G035500 DN AT1G66980.1 Suppressor of npr1-1 constitutive 4013G134900 DN AT4G02340.1 Alpha/beta-hydrolases superfamily protein010G155600 DN AT1G53440.1 Leucine-rich repeat transmembrane protein kinase016G011200 DN AT1G53440.1 Leucine-rich repeat transmembrane protein kinase017G035400 DN AT5G38260.1 Protein kinase superfamily proteinT114300 DN AT1G54470.2 RNI-like superfamily protein, encodes a Cf-like gene in Arabidopsis
that confers downy mildew resistance to several isolates ofPeronospora parasitica.
003G059700 DN AT1G15460.1 HCO3-transporter family006G007200 DN AT3G21790.1 UDP-glycosyltransferase superfamily protein015G104200 DN AT5G50915.1 Basic helix-loop-helix (bHLH) DNA-binding superfamily protein007G002500 DN AT1G78950.1 Terpenoid cyclases family protein007G141400 DN AT5G38280.1 PR5-like receptor kinase004G025900 DN AT4G05200.1 Cysteine-rich RLK (RECEPTOR-like protein kinase) 25001G430100 DN AT3G20340.1 Expression of the gene is down regulated in the presence of paraquat,
an inducer of photoxidative stress003G153700 DN NA
a Direction of regulation in the resistant clones (DN34 or NM6). Up: increase in expression; DN: decrease in expression; M: mixed regulation direction among the resistantclones and comparison pairs; NA: no match.
H. Liang et al. / Plant Science 223 (2014) 69–78 75
Table 5Common S. musiva loci identified in inoculated leaf samples among all four Populus hybrid clones.
Septoria protein ID GO annotation KOG annotation
150229 GO:0003676 Nucleic acid binding KOG0118 FOG: RRM domainGeneral function prediction only
KOG0023 Alcohol dehydrogenase, class VSecondary metabolites biosynthesis, transport and catabolism
148691 GO:0004096 catalase activity; GO:0006979 response to oxidativestress; GO:0006118 electron transport
KOG0047 Catalase inorganic ion transport and metabolism
135169 GO:0004768 stearoyl-CoA 9-desaturase activity; GO:0046914transition metal ion binding; GO:0020037 heme binding; GO:0016020membrane; GO:0006633 fatty acid biosynthetic process; GO:0005783endoplasmic reticulum; GO:0005506 iron ion binding; GO:0016491oxidoreductase activity; GO:0016717 oxidoreductase activity, actingon paired donors, with oxidation of a pair of donors resulting in thereduction of molecular oxygen to two molecules of water;GO:0006629 lipid metabolic process
KOG1600 Fatty acid desaturaseLipid transport and metabolism
150277 GO:0005515 protein binding None37996 GO:0005576 extracellular region KOG3017 Defense-related protein containing SCP domain
Function unknown150946 GO:0006118 electron transport; GO:0004601 peroxidase activity None128244 GO:0006118 electron transport; GO:0004601 peroxidase activity None151966 GO:0006120 mitochondrial electron transport, NADH to ubiquinone;
GO:0008137 NADH dehydrogenase (ubiquinone) activityKOG1687 NADH-ubiquinone oxidoreductase, NUFS7/PSST/20 kDasubunitEnergy production and conversion
KOG0857 60s ribosomal protein L10Translation, ribosomal structure and biogenesis
83520 GO:0006464 protein modification process KOG0001 Ubiquitin and ubiquitin-like proteinsGeneral function prediction onlyKOG0001 Ubiquitin and ubiquitin-like proteinsPosttranslational modification, protein turnover, chaperones
KOG0062 ATPase component of ABC transporters with duplicatedATPase domains/translation elongation factor EF-3bTranslation, ribosomal structure and biogenesisKOG0062 ATPase component of ABC transporters with duplicatedATPase domains/translation elongation factor EF-3bAmino acid transport and metabolism
146962 GO:0051444 negative regulation of ubiquitin-protein ligase activity;GO:0051443 positive regulation of ubiquitin-protein ligase activity;GO:0051440 regulation of ubiquitin-protein ligase activity during
rotein
KOG2806 ChitinaseCarbohydrate transport and metabolism
eroxidase activity) and involved in lipid transport/metabolismnd chitinase/carbohydrate transport/metabolism (Table 5).
iscussion
S. musiva is a major pathogen of Populus and can cause leafpot and stem canker diseases of susceptible clones. Comparedo other economically important pathogens, little is known abouthe molecular mechanisms underlying the Populus–Septoria inter-
ction. However, the genomes of S. musiva and P. trichocarpa,
host for the pathogen, have been sequenced. This provides anique opportunity to analyze this pathosystem. In this study, wepplied RNA-Seq, a high throughput and revolutionary tool for
ligase
transcriptomics, to identify pathogen responsive genes in the host.Overall, 78% of the 22.8 Gb of sequence generated were success-fully aligned to the P. trichocarpa genome, similar to what has beenreported in other RNA-Seq studies of plant tissues inoculated withpathogens: 50% in cotton (23), 69% in Arabidopsis (25), and 86% insoybean (24). The currently annotated Populus loci (41,291, phy-tozome v.9.0) were all represented with at least one aligned readin this study. The remaining 22% of the reads may be unique to thePopulus genotypes (Table 2) used in this study, which were differentspecies than P. trichocarpa. In addition, a total of 1392 putative new
areas of transcription in the P. trichocarpa genome were identified.
Comparisons of S. musiva-inoculated and mock-inoculatedleaves within the same clones resulted in only a few DE genes, ran-ging from 0 to 7 genes. All the DE genes had decreased expression
7 Scienc
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n inoculated leaves with the exception of 3 genes found inM6. Particularly in NC11505, a highly susceptible clone, all dif-
erentially expressed genes that had an annotation are diseaseesistance related (natural resistance-associated macrophage pro-ein 3, OBP3 (ocs element binding protein 3)-responsive gene 1, and-cysteine peroxiredoxin 1) and showed at least a 4-fold decrease ofxpression in inoculated leaves. In Arabidopsis, expression of OBP3-esponsive gene 1 was found to be upregulated by salicylic acidSA) and down regulated by jasmonic acid (JA) [38]. Peroxiredox-ns (Prx) are antioxidant enzymes. There is experimental evidenceor a triple Prx function in plant cell biology as: (i) antioxidant;ii) modulator of cell signaling pathways related to reactive oxygenpecies and reactive nitrogen species; and (iii) a redox sensor [39].hus, metabolic integrity, protection of DNA from oxidative dam-ge, and modulation of intracellular signaling were compromisedn NC11505 plants inoculated with the pathogen, which may haveontributed to greater disease severity.
When combining the data from both the resistant clones (DN34nd NM6), seven DE genes were identified when compared toontrol trees, two of these, encoding basic chitinase and Kunitzrypsin inhibitor, were responsive to wounding and insect elicitorsn Populus [40]. The Potri.010G007900 locus encodes a Kunitz fam-ly trypsin and protease inhibitor protein (KTI) (PtKTI A1, protein ID65533). Trypsin inhibitors are usually 20 kDa proteins with one orwo polypeptide chains that play important roles in plant defensegainst biotic stresses [41]. Ma et al. [42] identified thirty-one PtKTIroteins by searching the genome of P. trichocarpa. Their data sug-ested that Populus could deploy KTI genes actively and selectivelyn an insect-specific manner. Another locus Potri.002G014000ncodes a putative prolyl oligopeptidase, which belongs to theerine hydrolase family that has been suggested to play a roleuring the Botrytis cinerea–Arabidopsis interaction [43]. Kaschanit al. [43] revealed that prolyl oligopeptidase activity was down-egulated during Botrytis infection at 5 days post-infection. Itill be informative to investigate the physiological and molecu-
ar functions of these genes in detail. Potri.009G141800 encodes pathogenesis-related (PR)-3 protein (basic chitinase). Transgeniclants overexpressing basic chitinase have shown enhanced resis-ance to the necrotroph B. cinerea and the biotrophic powdery
ildew fungus [44]. One of the seven genes identified as differ-ntially expressed in the two resistant clones had no significantatch in any public database. The low number of DE genes obtained
n comparisons of Septoria-inoculated and mock-inoculated leavesay suggest that the majority of transcriptome changes might have
ccurred prior to four days post inoculation sampling time.There was an average of 84 DE transcripts in mock-treated
eaves among different clones. Compared to susceptible clones,he resistant clones DN34 and NM6 had higher expression levelsf disease/stress related genes. For instance, transcripts encod-ng sulfotransferase 16 (AT1G74100) and MLP-like protein 28AT1G70830) were more abundant in DN34 and NM6 than inC11505. Compared to other clones, DN34 was most resistantased on our current whole plant inoculation assay (Table 1) and
previous study with artificial inoculation of stems [8]. The DEenes found in DN34 were most enriched with oxidation–reductionunctions, which counteract oxidative stress. In particular, dis-ase/stress related proteins, such as leucine-rich repeat (LRR),hitinases (basic chitinase PR3 and chitinase-like protein 2),ermin-like proteins, beta glucosidase, glutathione S-transferase,isease resistance-responsive (dirigent-like protein) family pro-ein, terpene synthase, sulfotransferase 16, MLP-like protein8, and CAP (cysteine-rich secretory proteins, Antigen 5, and
athogenesis-related 1 protein) superfamily protein had a signif-
cantly higher transcript expression level in DN34. The GO termf oxidation–reduction functions were over-represented in the 31E genes common between the comparisons of resistant clones
e 223 (2014) 69–78
(DN34 and NM6) vs the highly susceptible clone NC11505 in themock-inoculated leaves. It is likely that these genes are constitu-tively expressed in the resistant clones, resulting in an immunesystem primed for counteracting pathogen attack prior to stress.
Except for the comparison between two susceptible clones,DN164 vs NC11505, the average number of DE genes greatlyincreased in leaves challenged with the pathogen when com-pared to mock-inoculated leaves (884 vs 84), indicating that thereare more differences between resistant and susceptible clones atthe transcriptomic level following fungal inoculation. The mock-inoculated leaves had 17 and 11 common DE genes between DN34vs susceptible clones (NC11505 and DN164) and NM6 vs suscep-tible clones, respectively. However, there were a greater numberof common DE genes, 332 (DN34 vs NC11505 and DN34 vs DN164)and 197 (NM6 vs NC11505 and NM6 vs DN164), found among inoc-ulated leaves. In addition, there were 37 common DE loci amongcomparisons of resistant clones vs susceptible clones, 36 of whichwere regulated in the same direction in both the resistant clones.These results indicate that these resistant clones are more effi-cient at inducing a transcriptomic response following pathogenperception and triggering a resistance response. This includes anincreased expression of a putative multi antimicrobial extrusionprotein gene. In contrast, no DE genes were identified from com-parisons between the two susceptible clones, DN164 vs NC11505,in Septoria-challenged leaves, indicating no significant transcrip-tomic differences four days after inoculation.
Ten of the 12 common DE loci involved in plant-type hyper-sensitive responses (GO:0009626) showed increased expression inthe susceptible clones. These loci correspond to 4 kinds of pro-teins: NB-ARC domain-containing disease resistance protein (4loci), phospholipase A 2A (3 loci), MutT/nudix family protein (1locus), and an elicitor-activated gene 3-1 product (2 loci) (Supple-mental Material 5). Resistance (R) proteins in plants are involved inpathogen recognition and subsequent activation of innate immuneresponses. Most of the R proteins contain nucleotide-binding site(NBS) and leucine-rich repeat (NBS-LRR) domains, which is part ofa larger entity called the NB-ARC domain. The NB-ARC (nucleotidebinding adaptor shared by NOD-LRR proteins, APAF-1, R proteinsand CED4) domain contains several defined motifs characteristicof “signal transduction ATPases with numerous domains” (STAND)family of ATPases [45]. Conformational changes caused by ATPhydrolysis are thought to regulate downstream signaling [46].A genome-wide analysis by Kohler and collaborators [47] hasrevealed 402 genes coding for NB-LRR proteins in P. trichocarpa.The MutT/nudix family protein negatively regulates the function ofR genes and programmed cell death in Arabidopsis [48].
Unlike interactions with biotrophic plant pathogens, whererecognition of a pathogen-produced effector by a host resistance(R) gene leads to resistance (effector triggered immunity; gene-for-gene model) [49,50], recognition of necrotophic plant pathogens bythe host leads to susceptibility (effector triggered susceptibility;inverse gene-for-gene model) ([49–51], and review in [52]). Ourdata are consistent with the inverse gene-for-gene model wheretranscripts involved in effectors-recognition were up-regulatedin susceptible genotypes resulting in disease. The loci encodingNB-ARC domain-containing disease resistance proteins will be val-idated in the future. It will be informative to compare the sequencesof these loci between resistant and susceptible clones. Recently,it has been reported that host-specific toxins (HSTs), also knownas necrotrophic effectors, induce cell death and promote diseasein host genotypes expressing a specific and often dominant sus-ceptibility gene [53]. These HSTs are thought to be critical for the
virulence of necrotrophic fungal pathogens and often target NB-ARC domain containing genes triggering disease development [53].However, necrotrophic effectors produced by S. musiva, potentiallytargeting these proteins, remain uncharacterized.
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H. Liang et al. / Plant
A second important pathogen of poplar is Melampsoraarici–populina, which is a fungal biotroph and causes leafust disease. In contrast to the poplar–Septoria system, theoplar–Melampsora system has been relatively well studied. For
nstance, an analysis of the pathogen’s genome has revealedhe presence of 1184 small secreted proteins (SSP)-encodingenes, representing candidate effectors [54]; and two NBS-LRR-ich regions (Rus and R1) have been identified in chromosome9 as qualitative and quantitative resistance loci [55]. As theoplar–Melampsora rust interaction is emerging as a model fortudies of tree immunity and fungal biotrophy, our efforts tonderstand the poplar–Septoria pathosystem will help advanceur understanding of necrotrophic pathogens.
It is also interesting to note that there was a significant posi-ive relationship detected between growth rate and susceptibilityR-squared = 0.34). This would seem to suggest the presence of atness cost associated with disease resistance. This assumption isased on the premise that there are limited resources available forlant growth. Adding disease resistance to the metabolic load of alant will limit the resources available for other activities. This mayesult in a decrease in growth and/or yield of the resistant geno-ype. This phenomenon has been documented in other systems56–58]. In order to fully understand and validate this relationship,urther work needs to be conducted across multiple years undereld conditions.
In conclusion, strong defense mechanisms involved inxidation–reduction, protein fate, secondary metabolism, andccumulation of defense-related gene products (such as antimi-robial proteins) may contribute to Septoria resistance in DN34nd NM6. Increased expression of the four loci encoding NB-ARComain-containing disease resistance proteins may contribute tohe host susceptibility in DN164 and NC11505 through interactionith effectors produced by the pathogen. The current findingsave provided the first insights into the transcriptomic responsef Populus leaves to S. musiva, a damaging fungal pathogen. Theifferentially expressed genes identified will be further studied forheir roles in plant–pathogen interactions.
cknowledgments
We thank Dr. Glen R. Stanosz for providing the hybrid poplaruttings, Dr. Shivegowda Thammannagowda for help collecting theeaf samples used in RNA-Seq, and Ruqian Qin for inoculating therees. This study was supported by the National Institute of Foodnd Agriculture, USDA (2011-67010-30197 to Liang, 2012-34103-9771 to LeBoldus, and SC-1700449 with a Clemson Universityxperiment Station technical contribution number of 6143).
ppendix A. Supplementary data
Supplementary data associated with this article can beound, in the online version, at http://dx.doi.org/10.1016/.plantsci.2014.03.004.
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