Responses of Highly Resistant and Susceptible Maize to Vascular Puncture Inoculation with Maize Dwarf Mosaic Virus

Physiological and Molecular Plant Pathology 86 (2014) 19e27

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Physiological and Molecular Plant Pathology

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Responses of highly resistant and susceptible maize to vascular puncture inoculation with Maize dwarf mosaic virus

Bryan J. Cassone a, Zhenbang Chen b,1, Joseph Chiera a,2, Lucy R. Stewart a,b, Margaret G. Redinbaugh a,b,* a USDA, ARS Corn, Soybean and Wheat Quality Research, Ohio Agriculture Research and Development Center (OARDC), Wooster, OH 44691, USA b Department of Plant Pathology, Ohio State University, OARDC, Wooster, OH 44691, USA article info abstract

Article history: Mechanisms for genetic resistance to diseases caused by potyviruses, especially Maize dwarf mosaic virus Accepted 24 January 2014 (MDMV), in maize (Zea mays L.) remain unclear. Previous studies indicated that MDMV can replicate in and spread to groups of cells in maize leaves of the virus resistant inbred line Pa405, but systemic Keywords: movement of the virus did not occur. In this study, the responses of Pa405 and the virus-susceptible inbred Maize line Oh28 were compared in germinating maize embryos. Four days post inoculation (dpi), MDMV was Potyvirus detected immunocytochemically in 45% of resistant and 19% of susceptible shoots, respectively. Systemic Gene expression movement of MDMV out of inoculated leaves occurred between 2 and 4 dpi in Oh28 seedlings. Microarray Resistance Microarrays analysis was used to examine the effects of MDMV inoculation on gene expression in shoots of resistant and susceptible lines at 4 dpi. Approximately 15% of transcripts were differentially expressed between the resistant and susceptible lines. Of the transcripts with >10 fold greater expression in one line, more than 70% were up-regulated in the resistant line. Substantially fewer genes were differentially expressed in MDMV- and mock-inoculated shoots, with 21 and 30 genes being differentially regulated in the susceptible and resistant lines, respectively. For a subset of genes, differential expression was validated using quantitative real-time PCR. There was no clustering of differentially expressed transcripts in the previously identiﬁed QTLs important for MDMV resistance. These results provide a short list of virus responsive transcripts to begin to explain the resistance response in Pa405 versus the susceptible response in Oh28. Published by Elsevier Ltd.

1. Introduction MDMV resistance [40], with genetic modifiers on chromosomes 3 and 10 [25]. The same, or closely linked, loci are important for Maize (Zea mays L.) resistance to viruses has been well- resistance to SCMV, WSMV and the potyviruses Johnsongrass characterized at the genetic level, and loci responsible for resis- mosaic virus (JGMV) and Sorghum mosaic virus [13,26,41,66,68,69], tance to viruses in the family Potyviridae including the potyviruses Redinbaugh and Pratt, 2008 [12,58]. Although fine mapping of Maize dwarf mosaic virus (MDMV), Sugarcane mosaic virus (SCMV) potyvirus resistance is advancing, especially resistance to SCMV and the tritimovirus Wheat streak mosaic virus (WSMV) have been derived from the European line FAP1360, genes for virus resistance mapped in a number of genotypes [46]. Resistance in the highly in maize remain to be isolated [21,59]. Susceptible maize lines, resistant maize inbred line Pa405 is among the best characterized including the inbred line Oh28, develop symptoms that include genetically. Here, a single dominant gene on chromosome 6 confers dwarfing, chlorotic spots, streaks and occasional necrotic lesions after inoculation with MDMV [25]. Naturally transmitted by aphids, MDMV can also be transmitted mechanically by several methods including leaf rub-inoculation and vascular puncture inoculation

* Corresponding author. USDA, ARS Corn, Soybean and Wheat Quality Research, (VPI) of germinating kernels [33,34]. MDMV levels in inoculated Ohio Agriculture Research and Development Center (OARDC), Wooster, OH 44691, leaves of the resistant line Pa405 were similar to those in suscep- USA. Tel.: þ1 330 263 3065; fax: þ1 330 263 3841. tible controls, and virus spread within inoculated leaves could be E-mail addresses: [email protected], [email protected] (M. detected serologically [30]. Similarly, when germinating kernels of G. Redinbaugh). Pa405 were inoculated with MDMV by vascular puncture inocula- 1 Current address: Department of Medicine, Biochemistry and Cancer Biology, Meharry Medical College, Nashville, TN, USA. tion, bright mosaic symptoms developed on leaves that were pre- 2 Current address: ArrayXpress, Inc., Raleigh, NC, USA. sent in the embryo at the time of inoculation, but no systemic

0885-5765/$ e see front matter Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pmpp.2014.01.002 20 B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27 infection occurred [35]. Together, these data indicate that MDMV embryo were excised from the germinating seed, then fixed and can replicate and move from cell-to-cell in leaves of resistant embedded with paraffin as described [18]. MDMV was detected plants, but that a barrier to systemic virus movement is present in immunocytochemically using polyconal antisera against MDMV- resistant plants. Although the ability to replicate and move locally OH [25]. For microarray analysis, the shoot portion of the germi- in resistant lines is similar to that described for the RTM-based nating embryo was excised from kernels, immediately frozen in resistance to Tobacco etch virus in Arabidopsis [37] and Rx-mediated liquid nitrogen, and stored at 80 C until RNA isolation. The resistance to Potato virus X in potatoes [3], molecular mechanisms experiment was replicated six times. associated with blocking systemic movement of MDMV in Pa405 To determine the time of MDMV systemic movement, the distal are not clear. 0.5 cm of primary leaves of 7 day old Oh28 seedlings were leaf rub- Viral pathogenesis is accompanied by a variety of changes in inoculated with purified MDMV inoculum prepared as for VPI, and plant gene expression that reflect the ways that viruses broadly and plants were transferred to a growth chamber at 25 C with a 12/12 h specifically interact with host cells for either defense or suscepti- light/dark cycle. The primary leaf was excised at the base imme- bility [17,20,29,39,62e64]. Most studies on gene expression in diately after inoculation and at 1, 2, 3, 4, 6 and 10 dpi. Systemic response to viruses have been performed in model dicotyledonous movement of MDMV was assessed as symptom development in hosts (primarily Arabidopsis and Nicotiana spp.) using compatible upper non-inoculated leaves at 14 dpi, and the presence of MDMV virus interactions; fewer studies have compared responses of was verified using a tissue blot immunoassay [25]. resistant and susceptible plants [22,38]. General plant responses to viruses include several pathways (reviewed in Whitham et al. [64]) 2.3. RNA isolation and cDNA synthesis including: 1) cellular stress/defense gene responses; 2) accumulation of viral proteins and heat shock proteins; 3) developmental Total RNA was isolated and purified from isolated shoots using a effects/hormone responses; and 4) activation of RNA silencing standard guanidine thiocyanate procedure [9]. The RNA pellet was responses. resuspended in water, extracted with phenol:chloroform, precipi- A body of studies on the responses of maize and rice to infection tated with Na-acetate and ethanol, and dissolved in diethylpyr- with viruses is beginning to emerge. As with model systems, most of ocarbonate (DEPC)-treated water. RNA quality and quantity were these studies are focused on compatible interactions [6,24,49e assessed using the NanoDrop ND-1000 Spectrophotometer 51,53,57] and few have compared expression in resistant and sus- (Thermo Scientific, Wilmington, DE) at 230 nm, 260 nm, and ceptible hosts [56,60]. Using microarrays, these studies showed only 280 nm. Integrity was further verified by running 10 mg samples on a small proportion of genes (<5%) respond differentially in resistant 1% denaturing agarose gels [48]. Total RNA (1 mg per sample) was and susceptible maize seedlings after inoculation with the potyvirus amplified and labeled with Cy3 or Cy5 (Amersham, GE Healthcare, Sugarcane mosaic virus for up to 24 h. Similarly, a limited number of Pittsburgh, PA) using the Amino Allyl MessageAmpÔ II aRNA proteins were differentially expressed in resistant and susceptible Amplification kit (Life Technologies, Grand Island, NY) according to lines 12 days after SCMV inoculation [67]. It is plausible that tran- the manufacturer’s recommendations. Labeled cDNA was purified scriptional responses predominately occur at more intermediate using RNeasy (Qiagen, Valencia, CA), and the amount of labeled time points and were consequently missed in these studies. RNA was determined from the absorbance at 550 nm and 650 nm The responses of resistant and susceptible maize to inoculation for Cy3 and Cy5, respectively. with MDMV during the period when systemic movement occurs remain poorly understood. Based on previous results by Ref. [30] 2.4. Microarray hybridization verified in this study, we predicted that systemic virus movement began between 2 and 6 days post inoculation (dpi). VPI of germi- Long oligonucleotide microarrays were obtained from the Maize nating maize kernels with potyviruses is highly efficient, with Oligonucleotide Microarray Project, and slides were prepared for many cells becoming infected in both the resistant and susceptible hybridization as described [15]. Slides were pre-hybridized in 5 maize [35]. Therefore, we compared global transcriptional re- SSC (1 SSC ¼ 15 mM Na-citrate, pH 7, 0.15 M NaCl) containing 0.1% sponses of the resistant (Pa405) and susceptible (Oh28) inbred lines SDS and 1 mg/ml BSA at 42 C for 45 min. Hybridization solution, at 4 days after VPI with MDMV using the microarrays designed by consisting of 16 ng/ml each Cy3 and Cy5 labeled antisense RNA in the Maize Oligonucleotide Array Project [15]. 5 SSC, 50% formamide, 0.1% sodium dodecyl sulfate, 0.4 mg/ml salmon sperm DNA and 0.4 mg/ml tRNA, was incubated at 95 C for 2. Materials and methods 2 min, then quenched in ice, prior to being applied to slides (185 ml/ slide). The solution was covered with a HybriSlip (Sigma, St. Louis), 2.1. Germplasm and virus maintenance and slides were incubated in a humid chamber at 42 C for 12 h. Slides were then washed at room temperature for 5 min each with: An Ohio isolate of MDMV was maintained by serial passage on 2 SSC containing 0.1% SDS; 0.1 SSC; and, twice with 0.05 SSC. susceptible maize as previously described [26,41]. Maize inbred Slides were spun dry at 2200 g for 4 min at room temperature lines Oh28 and Pa405 were maintained at the Ohio Agricultural then scanned using an Affymetrix (Santa Clara, CA) 428 Array Research and Development Center, Wooster, OH [33]. Scanner. Raw feature pixel intensity values for each probe were obtained using GenePix Pro 6.0.1.25 software (Molecular Devices, 2.2. Maize dwarf mosaic virus infection of maize Sunnyvale, CA). Three independent hybridizations, each including two of the biological replications, were done. For each hybridiza- For microscopy and microarray analysis, fifty kernels each of tion, probes for one biological replication were labeled with Cy3 Oh28 and Pa405 were inoculated with MDMV using VPI as previ- and those from the second replication with Cy5 (Fig. 1). ously described [34]. Virus inoculum consisted of 2 ml MDMV virions (20 mg protein/ml) in 0.01 M Tris-0.01 M Na-Citrate buffer, pH 2.5. Microarray data analysis 7 (TCB), purified as described [36]. For mock-inoculated samples, fifty kernels of each line were similarly inoculated with 2 ml of TCB. Raw pixel intensity values in GenePix Results (.gpr) files were Inoculated kernels were incubated at 30 C in the dark for 4 d. For first used to assess data quality. Probes that were poorly hybridized immunocytochemical detection of MDMV, the scutellum and using the flag quality index set to 100 and oligo probes with B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27 21

Expression levels were measured separately for reference and target genes, using three biological replicates for each maize line treatment combination. Relative transcript abundance was calcu- DDCt lated using the 2 method [32]. Threshold cycle (Ct) values reported by the CFX96 Real-Time PCR Detection System were normalized to the reference gene and converted to relative log2- Fig. 1. Experimental design for microarray experiments. Each arrow represents one fold differences between maize lines and treatments. One-tailed t- slide set. The arrow head indicates the treatment labeled with one dye (e.g., Cy5) and tests were used for statistical validation of differential expression, the tail the treatment labeled with the other dye (e.g., Cy3). The black and gray arrows under the hypothesis that signiﬁcant expression differences represent the two biological replications tested in the same experiment. In two ex- detected from the microarray would also be detected by RT-qPCR. periments, Cy5 was the dye used for the arrow head and Cy3 the tail. In the remaining experiment the dyes were reversed. RNA was isolated from the resistant line Oh28 (Sus.), or the resistant line Pa405 (Res.). 3. Results

3.1. Microarray hybridization missing intensity values were omitted from further analysis. The fi resulting les were then imported into Bioconductor [16], an open- At four days post-VPI, MDMV was detected in shoot tissues of source software project based on the R programming language both the resistant (Pa405) and susceptible (Oh28) maize inbred (www.r-project.org). Quantile normalization was accomplished lines (Fig. 2). At least some cells in 45% of resistant and 19% of using the normalize quantiles function [4]. All probe sequences susceptible plants were positive for MDMV in developing shoots, were individually mapped to the B73 AGPv2 assembly in Ensembl and more individual shoots were infected at 4 dpi than at 2 dpi [52] using BLASTn [1] to determine which gene (if any) they (Table 1). When similarly inoculated kernels were allowed to interrogated. Of the 57,542 probes represented on the arrays, develop into seedlings, 64% and 70% of resistant and susceptible < 5 38,099 mapped to 27,649 maize genes (E-value 10 ). The plants, respectively, developed mosaic symptoms on the primary discrepancy between number of probes and genes is because some leaf, indicating that the inoculation process was similarly efficient genes were interrogated by more than one probe. Functional in the two lines. The higher MDMV infection rates in the shoots and annotation of genes was updated based on gene ontologies (GO roots of the resistant line at 2 and 4 dpi was surprising given the terms) using BioMart software [27]. inoculation method was identical, and may be indicative of greater The subset of expressed genes was obtained using a method localized virus replication in Pa405. To identify the approximate similar to [7]: oligo probes corresponding to expressed genes were period when systemic virus movement occurred in the susceptible fi de ned as those whose GenPix Pro hybridization signals exceeded line, the distal 0.5 cm of the primary leaf of Oh28 seedlings were 500 for at least three of the six line treatment replicates. inoculated with MDMV, and the leaf then was excised at the base fi Expressed probes interrogating the same gene, as de ned above, either immediately (0 dpi) or at 1 to 10 dpi (Table 2). Systemic virus were collapsed into a single value by taking the mean normalized movement, assessed by development of symptoms on upper non- probe intensity. Files containing the raw (GPR) and normalized data inoculated leaves (i.e. the fourth leaf or later), was not detected have been deposited with Array Express in MAGE-TAB format and when the leaf was excised at 0 or 1 dpi, but occurred in about 35% of are available under Accession no. E-MTAB-1485. seedlings by 2 dpi and 82% of seedlings by 4 dpi. These data sug- Differential gene expression was evaluated using the two-way gested that systemic movement of MDMV occurred between 2 and analysis of variance (ANOVA) model in the nlme package in R. 5 dpi in the susceptible line, and we hypothesized that this time Inbred line, treatment and their interaction were used as the main frame was similarly important for the expression of resistance in factors, and replicate as a random factor. Genes were considered to be differentially expressed if P < 0.01, unless otherwise specified. Subsequently, Bayes moderated t-tests were carried out using the limma package in R to identify the sets of virus responsive genes within each maize line. Similarly, transcriptome variation between the two maize lines was also examined.

2.6. Quantitative RT-PCR

DNase treated total RNA (3 mg per sample) used in the microarray experiments were used for quantitative RT-PCR (RT-qPCR). Single-stranded cDNA was synthesized using the iScript kit (Bio- Rad, Hercules, CA) per the manufacturer’s instructions. Primers for targeted mRNAs were designed using Primer3 [47] and primer sequences are found in Table S1. In preliminary experiments, 50 nmol, 300 nmol, and 900 nmol of primer were tested determine optimal qPCR conditions for each gene. Actin-1 (MZ00038005) was used as a reference gene. qPCR reactions (15 ml) were performed in triplicate using IQÔ 2 SYBR Green Supermix (Bio-Rad) and 300 nmol of each primer on a CFX96 Real-Time PCR Detection System (Bio-Rad) according to the manufacturer’s recommendations. Cycling conditions were: 50 C for 2 min; 95 C for 10 min; 41 Fig. 2. Detection of MDMV in resistant and susceptible maize at 4 days post vascular cycles of denaturation at 95 C for 15 s, and annealing/extension at puncture inoculation. Embryos were excised from inoculated resistant (Pa405) and susceptible (Oh28) plants at 4 dpi. After ﬁxing and sectioning tissues, MDMV was 60 C for 1 min; and, 95 C for 15 s. PCR efﬁciency (E) was evaluated detected using immunocytochemical detection with MDMV antisera as described by performing qPCR with serial dilutions of cDNA and target gene (Greyson et al., 1996). The scale bar for each image represents 1 mm. The arrows (1/slope) primers and the equation E ¼ 10 [43]. indicate the presence of MDMV coat protein (purple staining). 22 B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27

Table 1 identify differential accumulation of transcripts between the Maize infection after vascular puncture inoculation with Maize dwarf mosaic virus. resistant (Pa405) and susceptible (Oh28) lines. More than 15% of Tissue dpia Resistantb %Inf Susceptible %Inf transcripts (2580) had greater than a two-fold difference in accu- < Shootc 22616 mulation between the lines (P 0.01), with about half (1336) 44519 accumulating to higher levels in the susceptible line. There were Root 2 42 18 222 with more than a four-fold difference and 52 with more than a 45024 ten-fold difference in accumulation between the two lines. Of the Primary Leaf 10 64 70 transcripts with more than ten-fold greater accumulation in one a DPI, days post inoculation. line, more than 70% were up-regulated in the resistant line. b Percentage infection in resistant (Pa405) and susceptible (Oh28) plants. c Genome locations and putative gene models associated with these For shoots and roots, the percentage of infected tissues was determined using fi immunocytochemical detection with MDMV antisera [18]. Primary leaves were transcripts were identi ed using POPCORN BLAST [5] against the considered positive if they developed mosaic symptoms at 8 days post inoculation. B73 RefGen_v2 (MGSC) maize genome assembly [52] (Table S2). Of Data are the mean for at least three independent experiments. the 52 transcripts, 36 of the corresponding probes aligned to at least one site in the maize genome with more than 85% identity, the resistant line. As expected, MDMV symptoms were never and function or functional domains could be predicted for 29 of the detected on upper non-inoculated leaves of the resistant line, encoded proteins. Eleven of the differentially regulated transcripts indicating systemic movement did not occur. encoded stress responsive genes and eight encoded transcription To characterize the molecular responses of maize to MDMV factors/regulators. inoculation, gene expression in MDMV- and mock-inoculated resistant (Pa405) and susceptible (Oh28) maize shoots was evalu- 3.3. Maize transcripts accumulating in response to MDMV ated at 4 dpi using microarrays from the Maize Oligonucleotide inoculation Array Project (Gardinger et al., 2005) (Fig. 1). In three independent experiments, six biological replications were hybridized on two- A total of 51 transcripts were identified as differentially color arrays of 57,542 oligo probes. Data from two samples on expressed in MDMV-inoculated vs. mock-inoculated shoots inde- one slide (one mock-inoculated Oh28 and one mock-inoculated pendent of maize line using Bayes-moderated t-tests, and 75% of Pa405 sample) were omitted from the analysis due to poor hy- the transcripts were up-regulated in the virus-inoculated treat- bridization quality. For the remaining hybridizations, 10,797 probes ment (Table S3). Eight transcripts had protein domains indicative of not interrogating genes in the AGPv2 maize genome assembly, with transcription factors, with six of these being up-regulated in missing expression values, or with low quality hybridization were MDMV-inoculated plants. Similarly, 21 and 31 transcripts accu- also eliminated from the analysis, as were an additional 13,134 mulated differentially (P < 0.01) between the mock- and MDMV- probes with low expression in both maize lines and both treat- inoculated treatments of the resistant and susceptible lines, ments. Of the remaining 33,611 expressed probes, 24,763 mapped respectively, with the majority of differentially expressed tran- to 22,133 maize genes in the AGPv2 filtered set (E-value < 10 5), scripts being up-regulated in MDMV-inoculated treatment (Fig. 3; and 12 interrogated the MDMV genome. For subsequent discussion, Tables 3 and 4, S4 and S5). Only one transcript was differentially a ‘transcript’ is defined as the signal associated with a specific gene expressed between treatments for both the resistant and suscep- on the microarray, and transcripts accumulating to higher levels tible lines (GRMZM2G479243). under a given set of conditions are referred to as ‘up-regulated’; e.g., Two-way ANOVA was used to identify the set of genes with a transcripts with higher levels in Pa405 relative to Oh28 will be significant treatment (MDMV- or mock-inoculated) maize line referred to as up-regulated in Pa405, and those with higher levels in interaction. Only three transcripts were identified that met this Oh28 than Pa405 will be referred to as up-regulated in Oh28. As criteria: GRMZM2G179301 (up-regulated in MDMV-inoculated expected, transcript levels for MDMV assessed by three and five Pa405), GRMZM5G830329 (up-regulated in mock-inoculated probes were significantly higher in the virus-inoculated treatments Pa405), and AC217910.3 (up-regulated in MDMV-inoculated of Pa405 and Oh28, respectively (data not shown). Oh28) (Table 5).

3.2. Differential transcript accumulation in mock-inoculated 3.4. Genomic distribution of MDMV responsive genes susceptible and resistant maize Sliding window analyses were used to assess whether the To examine basal transcriptome variation between the two differentially expressed transcripts disproportionately mapped to maize lines, a Bayes-moderated t-test (P < 0.01) was carried out to

Table 2 Systemic movement of MDMV in maize seedlings of the susceptible line Oh 28. Virus inoculum consisted of 2 ml MDMV virions (20 mg protein/ml) in 0.01 M Tris-0.01 M NaCitrate buffer, pH 7. Systemic movement was assessed as symptom development in upper non-inoculated leaves at 14 dpi, and the presence of MDMV was veriﬁed using a tissue blot immunoassay.

dpi 1a 2 3 Total

0 0/6 0/6 0/6 0/18 1 0/6 0/6 0/6 0/18 2 4/5 0/6 2/6 6/17 3 5/6 2/5 3/6 10/17 4 3/6 5/5 6/6 14/17 Fig. 3. Effect of MDMV inoculation on gene expression in resistant and susceptible 6 6/6 4/5 5/6 15/17 maize. The volcano plot shows relative expression difference for transcripts in 10 6/6 4/5 6/6 16/17 MDMV ¼ inoculated/mock inoculated resistant Pa405 gray circles and susceptible a Data presented are the number of symptomatic plants/total plants in inde- (Oh28 black triangles). The dotted line indicates P ¼ 0.01, with data points above this pendent experiments 1, 2 and 3. line being signiﬁcantly differentially expressed. B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27 23

Table 3 Differentially expressed transcripts in virus-inoculated and mock-inoculated resistant maize (Pa405).

Pa Log 2 FCb Trtmtc Gramene IDd Annotatione

0.0005 2.40 MDMV GRMZM2G479243f Brassinosteroid insensitive receptor kinase GRMZM2G479260 Hsp20/alpha-crystallin family protein 0.0025 1.13 MDMV GRMZM2G151536 F-box domain and LRR containing protein 0.0036 1.28 MDMV GRMZM2G060554 Remorin, C-terminal region 0.0039 1.30 MDMV GRMZM2G056039 Hsp70 protein 0.0051 1.21 MDMV GRMZM2G170602 Erythronate-4-phosphate dehydrogenase 0.0060 1.25 MDMV GRMZM2G135588 Citrate synthase 0.0065 1.33 MDMV GRMZM2G179301 Endo-1,3; 1,4-beta-D-glucanase 0.0078 1.18 MDMV GRMZM2G081729 Glycosyl hydrolase, family 43 0.0078 1.26 MDMV GRMZM5G815894 Ribosomal L18p/L5e 0.0085 1.33 MDMV GRMZM2G099253 DEAD/DEAH box helicase 0.0090 1.24 MDMV GRMZM2G078757 Major facilitator superfamily transporter 0.0091 1.17 MDMV AC226373.2 Zinc ﬁnger protein (C-x8-C-x5-C-x3-H type) 0.0092 1.21 MDMV GRMZM2G156713 sufB/sufD domain containing protein 0.0099 1.15 MDMV GRMZM2G045366 Ankyrin repeat/protein kinase 0.0016 1.28 Mock GRMZM2G156543 Protein phosphatase 2C 0.0043 1.38 Mock GRMZM2G098696 Heat shock transcription factor 0.0058 1.14 Mock GRMZM2G151909 VQ motif 0.0063 1.12 Mock GRMZM2G138423 Mitochondrial carrier protein 0.0066 1.13 Mock GRMZM2G171807 Protein tyrosine kinase 0.0077 1.22 Mock GRMZM5G830329 Cytochrome P450 0.0088 1.42 Mock GRMZM2G028432 ATP synthase subunit C

a P value for the mean difference in expression for six independent experiments. b Log 2 of the fold change in expression for six independent experiments. c Trtmt, Treatment; the treatment having higher expression of the transcript, MDMV-inoculated or mock-inoculated Pa405 plants. d Gene model record number for hits based on oligonucleotide probes identified by POPCORN BLAST. e Annotation from PFAM or best hit to the Arabidopsis or rice genomes based on POPCORN BLAST of oligonucleotide probe sequences. More detail is available in Table S4. f The region of the genome with highest identity to probe MZ00032788 encodes two putative genes, GRMZM2G479243 and GRMZM2G479260. loci associated with resistance. For this, we used the subsets of 212 efficiency of 1.95 (data not shown). The two probed portions of the and 270 transcripts that were differentially expressed between MDMV were similarly up-regulated in MDMV-inoculated Pa405 treatments at P < 0.05 in the resistant and susceptible lines, and Oh28 on both the microarray and RT-qPCR analyses (Fig. 4). The respectively. Resistance has been mapped to genomics regions GRMZM2G479243 transcript was identified as significantly up- roughly estimated from QTL-based mapping studies at w49 Mb for regulated in both maize lines after MDMV inoculation (P < 0.01). the chromosome 6 resistance locus (246 expressed transcripts In the RT-qPCR analysis, this transcript accumulated to higher levels derived from the microarray); w101 Mb for the chomosome 3 in MDMV-inoculated Oh28 (P < 0.05) and Pa405 (P < 0.001) rela- resistance locus (558 expressed transcripts); and 51 Mb for the tive to mock-inoculated plants, consistent with the microarray re- chromosome 10 resistance locus (531 expressed transcripts) [26]. sults. GRMZM2G180847 transcripts were up-regulated in MDMV- Using a chi-squared test, the observed numbers of differentially inoculated plants (P < 0.01) on the microarray analysis; however, expressed transcripts found within and outside the boundaries of it was not significantly up-regulated in MDMV-inoculated Pa405 or one or all three resistance loci were compared to the numbers Oh28 individually (Tables S4 and S5). Similarly, no differences be- expected in the two partitions, given the total number of expressed tween transcript levels in mock- or MDMV-inoculated Pa405 were genes and the length of each partition. Across both maize lines and seen for GRMZM2G180847 by RT-qPCR. These data indicate that considering all three loci together, there was not a significant genes identified as significantly differentially expressed on the overrepresentation of differentially expressed transcripts in the microarray analysis at P < 0.01 are likely to be similarly identified as resistance QTLs [Pa405: 12 transcripts, c2 ¼ 0.074 (1), P ¼ 0.785; differentially expressed by RT-qPCR. Oh28: 16 transcripts, c2 ¼ 0.016 (1), P ¼ 0.901]. Additionally, no differentially expressed transcripts were identified in the fine- 4. Discussion mapped w59 Kb region of chromosome 6 believed to contain a dominant gene important for resistance to Sugarcane mosaic virus We examined differential expression of maize transcripts in [59]. developing shoots of susceptible (Oh28) and resistant (Pa405) maize lines after VPI of germinating kernels with the potyvirus, 3.5. Quantitative real time PCR MDMV. We chose VPI of seeds as the inoculation method, because the virus is introduced into many more cells relative to leaf rub Differential expression of selected transcripts identified in the inoculation [35], which could increase our ability to discern microarray analysis was validated using RT-qPCR. Three RNAs were changes induced by the virus. Because potyvirus resistance in targeted for validation, including transcripts for GRMZM2G180847 maize is associated with prevention of systemic virus movement, (Table 3 and S4; similar to transcription factor RF2a), and both previous and current results indicate that systemic GRMZM2G479243 (Tables 3 and 4, S4 and S5; similar to Brassi- movement of potyviruses in susceptible lines occurs between 2 and nosteroid insensitive 1-associated receptor kinase 1), and the 5 dpi (Table S2) [8,30], we compared transcript accumulation in MDMV genomic RNA. Four sets of primer pairs were developed mock- and MDMV-inoculated maize inbred lines at 4 dpi. Rapid (two pairs probed different regions of the MDMV genome) with (less than 24 h) and longer-term (more than 12 days) responses of PCR efficiencies (E values) between 1.7 and 2.15 (Table S1). Maize resistant and susceptible maize to virus infection with SCMV actin1 was selected as the reference transcript, because it was examined previously using European and Asian germplasm [6,54e expressed at constant levels across all samples and had PCR 56,60]. We expect that timing of differential expression may be 24 B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27

Table 4 Differentially expressed transcripts in virus-inoculated and mock-inoculated susceptible maize (Oh28).

Pa Log2 FCb Trtmtc Gramene IDd Annotatione

0.0007 1.20 MDMV GRMZM2G465444 Pentatricopeptide repeat (PPR) superfamily protein 0.0018 1.29 MDMV GRMZM5G843302 Major facilitator superfamily 0.0022 1.13 MDMV GRMZM2G104047 Transposon protein 0.0023 1.51 MDMV GRMZM2G091857 NHf 0.0024 1.12 MDMV GRMZM2G099382 Citrate transporter 0.0038 1.16 MDMV GRMZM2G171595 Remorin, C-terminal region 0.0040 1.38 MDMV GRMZM2G050027 NH 0.0043 1.16 MDMV GRMZM2G481843 3-oxo-5-alpha-steroid 4-dehydrogenase 0.0047 1.15 MDMV GRMZM2G397402 Cytochrome P450 0.0057 1.19 MDMV GRMZM2G030823 F-box, LRR and FBD domain containing protein 0.0060 1.60 MDMV GRMZM2G114036 Patatin-like phospholipase 0.0065 1.28 MDMV GRMZM2G447617 Triose-phosphate Transporter 0.0066 1.14 MDMV GRMZM2G108084 Zinc ﬁnger protein, C3HC4 type 0.0067 2.00 MDMV GRMZM2G479243g Brassinosteroid insensitive receptor kinase GRMZM2G479260 Hsp20/alpha-crystallin family 0.0071 1.18 MDMV GRMZM2G096591 Glycosyl hydrolase, family 17 0.0071 1.18 MDMV GRMZM2G096591 Expansin precursor 0.0084 1.12 MDMV GRMZM2G152436 JAB1/Mov34/MPN/PAD-1 ubiquitin protease 0.0089 1.20 MDMV GRMZM2G177510 Glycosyl hydrolase, family 17 0.0090 1.15 MDMV GRMZM2G046459 Glycosyl hydrolase, family 17 0.0090 1.17 MDMV GRMZM2G132880 Phosphatidylethanolamine-binding protein 0.0094 1.22 MDMV GRMZM2G393897 FAE1/Type III polyketide synthase-like protein 0.0093 1.19 MDMV GRMZM2G407825 phospholipid-transporting ATPase 0.0095 1.20 MDMV GRMZM2G126367 Esterase 0.0099 1.41 MDMV GRMZM2G084694 Neutral/alkaline invertase 0.0100 1.27 MDMV GRMZM2G394060 NH 0.0036 1.37 Mock GRMZM2G103595 DVL family protein 0.0015 1.21 Mock GRMZM2G143142 NH 0.0026 1.17 Mock GRMZM2G056038 NH 0.0045 1.13 Mock GRMZM2G108416 Aminotransferase class IV 0.0063 1.38 Mock GRMZM2G380414 Psb(X) 0.0098 1.87 Mock GRMZM2G386674 Protein of unknown function DUF260

Table 5 Transcripts derived from the two-way analysis of variance (ANOVA) with signiﬁcant treatment (MDMV- or mock-inoculated) maize line (Pa405 or Oh28) interaction.

Oligo IDa Pb Gene IDc Chrd Start (nt) End (nt) Similarity toe

MZ00014548 0.001 GRMZM2G179301 6 14,508,7390 145,092,573 Os05g33100.1: endo-1,3; 1,4-beta-D-glucanase precursor MZ00020971 0.003 GRMZM5G830329 10 84,608,950 84,610,855 Os08g03682.1: Cytochrome P450; ferulate 5-hydroxylase MZ00030813 0.005 AC217910.3 1 217,273,728 217,280,603 AT3G13850: LOB domain containing protein 22; Os08g31080 DUF260

a Oligo ID, ID number for the probe. b P value for the mean difference in expression for six independent experiments. c Gene model record number for hits based on oligonucleotide probes identiﬁed by POPCORN BLAST. d Chr, chromosome. e Rice or Arabidoposis gene with the highest similarity to the gene. B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27 25

20 infected maize [24]. In infected susceptible Oh28, putative cyto- *** chrome P450, remorin, phosphatidylethanolamine-binding pro- *** tein, and polyketide synthase-like protein coding transcripts were *** 15 up-regulated, in addition to transcripts with putative roles in transcription, metabolism, and signal transduction. In Pa405, a *** different remorin-like transcript was detected as up-regulated in MDMV infected tissue, as well as transcripts encoding a putative 10 Hsp70, an LRR- and F-box containing protein similar to Arabidopsis COI1, a callose synthase-like protein (among three genes with significant treatment line interaction, Table 5), and a putative 5 tyrosine kinase. Transcripts for a phosphatase, two protein kinases Relative Expression including an OsWAK1 homolog identified in the RBSDV study [24], NS NS * *** and a cytochrome P450 were among those down-regulated in 0 infected relative to mock-inoculated resistant tissue (Table 3 and S4). Infection responsive transcripts included several that were previously implicated in the virus response (Tables 3e5 and S3e S5). Cytochrome P450 transcripts (matching two different genes and detected by two different oligo probes) were up-regulated in mock-inoculated Pa405 and MDMV-inoculated Oh28. Homologs of Treatment the cytochrome P450 transcript up-regualated in mock-inoculated maize kernels were previously implicated in stress responses Fig. 4. Verification of differential transcript accumulation at four days post inoculation including infection with Magnaporthe grisea, and Rice yellow mottle by RT-qPCR analysis. Levels of two Maize dwarf mosaic virus genome segments (MDMV virus [65]. A callose synthase (beta 1,3-glucanases) are upregulated, I and MDMV II) and two maize transcripts (GRMZM2G479243 and GRMZM2G180847) as reported in Arabidopsis [64] and rice [24]. Also in MDMV- relative to maize actin1 are shown. The average fold change for mock- (light stipple) and MDMV-inoculated (dark stipple) Oh28 and mock- (light stripe) and MDMV- inoculated Oh28 plants, a retrotransposon protein is up- inoculated (dark stripe) Pa405 are shown. Significant differences in transcript accu- regulated, perhaps in a generalized stress response [42]. mulation between mock- and MDMV-inoculated plants of the same genotype are A number of transcripts with putative functions in protein fi < < < indicated as NS, not signi cant; *P 0.05; **P 0.01; ***P 0.001. synthesis and degradation were identified in all four contrasts. In particular, a heat shock protein (Hsp) 70 was up-regulated in MDMV-inoculated Pa405. Hsp70s have many functions in cells, previous studies with SCMV [56,60]. This lack of clustering was also including involvement in the unfolded protein response of cells and observed for the 52 transcripts with more than 10-fold differences are triggered by high protein expression in cells or accumulation of in accumulation, although genes with potential functions virus misfolded proteins [2]. The RTM2 gene for Arabidopsis resistance to infection and pathogen response were identified (Table S2). the potyvirus Tobacco etch virus encodes an involved unusual Comparing MDMV-inoculated versus non-inoculated plants for Hsp20/a-crystallin protein [61], and transcripts encoding Hsp20/a- both genotypes together indicated that far fewer transcripts were crystallin homologs were up-regulated in virus-inoculated and differentially regulated according to treatment than genotype. The Oh28 plants. Somewhat surprisingly, a homolog of Arabidopsis heat 51 differentially expressed transcripts that were identified seems shock transcription factor B2A was down-regulated in MDMV- low but was comparable to other microarray studies examining the inoculated Pa405 plants, whereas heat shock proteins are differential response of resistance and susceptible maize seedlings commonly up-regulated in response to viral infection [64]. Protein to inoculation with the fellow potyvirus, Sugarcane mosaic virus degradation pathway proteins are differentially regulated, with a [55,60]. Of the differentially regulated transcripts, two annotated as COI1 homolog being very highly expressed in Pa405, and a ubiq- potentially involved in virus resistance, pathogen response, or uitin protease being up-regulated in MDMV-inoculated Oh28. A stress response were up-regulated in MDMV-inoculated tissues: an COI1 encodes an F-box containing protein that is involved in plant Hsp20/alpha crystalline-like gene and a cytochrome P450 resistance and susceptibility responses to viral, bacterial and fungal (Table S3). Further, nine transcripts with putative involvement in diseases [28,31]. Although no specific homologs of Arabidopsis signal transduction were identified as up-regulated in MDMV- genes required for RNAi were identified, transcripts implicated in inoculated relative to mock-inoculated plants. A homolog of RF2a, RNA processing and metabolism were found, including a DEAD-box a rice transcription factor that regulates the expression of the RTSV helicase that was up-regulated in Pa405. promoter and confers resistance to Rice tungro bacilliform virus Differential expression of transcripts for several genes previ- [10,11], was up-regulated in MDMV-inoculated maize. Transcripts ously implicated in plasma membrane raft structures was identified with putative involvement in transcription, transcript processing, in this study. Infection-responsive up-regulation of transcripts metabolism, and membrane trafficking were also detected as up- encoding major facilitator superfamily proteins and remorins were regulated in virus-inoculated tissue and may reflect either virus- was identified in both Oh28 and Pa405. Remorins are proteins aiding or virus-defensive regulation. present in membrane rafts and plasmodesmata that are proposed We expected that comparing virus- and mock-inoculated tran- to have important roles in hostemicrobe interactions [14,23], and a script regulation by genotype would provide insight into the remorin is involved in inhibiting Potato virus X (PVX) movement in mechanisms of resistance in Pa405. Surprisingly few transcripts (21 Solanaceous plants [44]. Two different remorin transcripts, both for Pa405 and 31 for Oh28) were detected as differentially encoding group 6 remorins [45], were up-regulated in MDMV- expressed between treatments. Only one is shared between geno- inoculated Pa405 and Oh28. Yet another remorin protein accumu- types, matching both a leucine rich repeat (LRR) receptor protein lated in the virus-susceptible line Mo17, but not in the resistant kinase (brassinosteroid-insensitive 1 in rice) and an Hsp20/alpha maize inbred line Siyi inoculated with SCMV [67]. crystalline-like gene (Tables 3 and 4, S4 and S5). This gene is also Although only a small proportion of transcripts responded up-regulated at 9e12 dpi in Rice black streaked dwarf virus (RBSDV) differently in resistant and susceptible maize seedlings after MDMV 26 B.J. Cassone et al. / Physiological and Molecular Plant Pathology 86 (2014) 19e27 inoculation, this is in line with previous findings from microarray [2] Aparicio F, Thomas CL, Lederer C, Niu Y, Wang DW, Maule AJ. Virus induction fl studies of maize after inoculation with the potyvirus Sugarcane of heat shock protein 70 re ects a general response to protein accumulation in the plant cytosol. Plant Physiol 2005;138:529e36. mosaic virus (SCMV) for up to 24 h [56,60]. No overlap was detected [3] Bendahmane A, Kanyuka K, Baulcombe DC. The Rx gene from potato controls among the lists of differentially expressed transcripts derived from separate virus resistance and cell death responses. Plant Cell 1999;11:781e91. our study and the SCMV studies. Possible factors contributing to the [4] Bolstad BM, Irizarry RA, Astrand M, Speed TP. A comparison of normalization methods for high density oligonucleotide array data based on variance and differences between transcript lists may be: probes on our array do bias. Bioinformatics 2003;19:185e93. not interrogate the same genes as the custom SCMV arrays; tech- [5] Cannon EKS, Birkett SM, Braun BL, Kodavali S, Jennewein DL, Yilmaz A, et al. nical differences between platforms including version of maize POPcorn: an online resource providing access to distributed and diverse maize project data. Int J Plant Genomics 2011;2011. Article ID 923035. genome assembly; differences in statistical methods, analytical [6] Cao YY, Shi Y, Li YQ, Cheng YQ, Zhou T, Fan ZF. Possible involvement of maize approach, and data interpretation; differences in life stage and time Rop1 in the defence responses of plants to viral infection. Mol Plant Pathol points analyzed; differences in virus inoculation methods; and 2012;13:732e43. [7] Cassone BJ, Mouline K, Hahn MW, White BJ, Pombi M, Simard F, et al. Dif- intrinsic differences in the responses of maize to MDMV and SCMV ferential gene expression in incipient species of Anopheles gambiae. Mol Ecol inoculation. 2008;17:2491e504. Based on the work described here, we have a relatively short list [8] Chaves-Bedoya G, Espejel F, Alcala-Briseno RI, Hernandez-Vela J, Silva- of virus responsive transcripts to begin to explain the resistance Rosales L. Short distance movement of genomic negative strands in a host and nonhost for sugarcane mosaic virus (SCMV). Virol J 2011;8:15. response in Pa405 versus the susceptible response in Oh28. The [9] Chirgwin JM, Arzybyla AE, MacDonald RJ, Rutter WJ. Isolation of biologically genetic background of these lines is different, so there are many active RNA from sources enriched in ribonuclease. 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Dev Genet 1996;18:244e53. nation in all its programs and activities on the basis of race, color, [19] Hansey CN, Vaillancourt B, Sekhon RS, de Leon N, Kaeppler SM, Buell CR. national origin, age, disability, and where applicable, sex, marital Maize (Zea mays L.) genome diversity as revealed by RNA-sequencing. PLoS status, familial status, parental status, religion, sexual orientation, One 2012;7:e33071. genetic information, political beliefs, reprisal, or because all or part [20] Huang ZL, Yeakley JM, Garcia EW, Holdridge JD, Fan JB, Whitham SA. Salicylic acid-dependent expression of host genes in compatible Arabidopsis-virus in- of an individual’s income is derived from any public assistance teractions. Plant Physiol 2005;137:1147e59. program. (Not all prohibited bases apply to all programs.) Persons [21] Ingvardsen CR, Xing YZ, Frei UK, Lubberstedt T. Genetic and physical fine with disabilities who require alternative means for communica- mapping of Scmv2, a potyvirus resistance gene in maize. Theor Appl Genet 2010;120:1621e34. tion of program information (Braille, large print, audiotape, etc.) [22] Ishihara T, Sakurai N, Sekine KT, Hase S, Ikegami M, Shibata D, et al. should contact USDA’s TARGET Center at (202) 720-2600 (voice Comparative analysis of expressed sequence tags in resistant and susceptible and TDD). To file a complaint of discrimination, write to USDA, ecotypes of Arabidopsis thaliana infected with cucumber mosaic virus. Plant e fi Cell Physiol 2004;45:470 80. Director, Of ce of Civil Rights, 1400 Independence Avenue, S.W., [23] Jarsch IK, Ott T. Perspectives on remorin proteins, membrane rafts, and their Washington, D.C. 20250-9410, or call (800) 795-3272 (voice) or role during plant-microbe interactions. Mol Plant-Microbe Interact 2012;24: (202) 720-6382 (TDD). USDA is an equal opportunity provider and 7e12. 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