Abstract
Background
The potyviruses sugarcane mosaic virus (SCMV) and maize dwarf mosaic virus (MDMV) are major pathogens of maize worldwide. Two loci, Scmv1 and Scmv2, have ealier been shown to confer complete resistance to SCMV. Custom-made microarrays containing previously identified SCMV resistance candidate genes and resistance gene analogs were utilised to investigate and validate gene expression and expression patterns of isogenic lines under pathogen infection in order to obtain information about the molecular mechanisms involved in maize-potyvirus interactions.
Results
By employing time course microarray experiments we identified 68 significantly differentially expressed sequences within the different time points. The majority of differentially expressed genes differed between the near-isogenic line carrying Scmv1 resistance locus at chromosome 6 and the other isogenic lines. Most differentially expressed genes in the SCMV experiment (75%) were identified one hour after virus inoculation, and about one quarter at multiple time points. Furthermore, most of the identified mapped genes were localised outside the Scmv QTL regions. Annotation revealed differential expression of promising pathogenesis-related candidate genes, validated by qRT-PCR, coding for metallothionein-like protein, S-adenosylmethionine synthetase, germin-like protein or 26S ribosomal RNA.
Conclusion
Our study identified putative candidate genes and gene expression patterns related to resistance to SCMV. Moreover, our findings support the effectiveness and reliability of the combination of different expression profiling approaches for the identification and validation of candidate genes. Genes identified in this study represent possible future targets for manipulation of SCMV resistance in maize.
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Background
SCMV and MDMV are positive-sense single strand RNA potyviruses that cause significant yield loss in susceptible genotypes of maize, sugarcane, and sorghum [1, 2]. SCMV is notably harmful in Europe and China, MDMV in the southern US Corn Belt [3]. Both closely related potyviruses are transmitted in a non-persistent manner by aphids mainly to members of the Poaceae family [4]. Disease symptoms are mosaic, chlorosis, leaf reddening, necrosis, and stunting [2, 5]. Both viruses spread systemically and particularly fast in young susceptible plants [6].
Out of 122 early-maturing maize dent inbred lines investigated by Kuntze et al. [7], three (D21, D32, and FAP1360A) were found to be completely resistant to SCMV, MDMV, JGMV, and SrMV, both in field and greenhouse experiments. Depending on the population used, one to five genes were assumed to be required for complete SCMV or MDMV resistance [3, 8–11]. Two major SCMV resistance genes, Scmv1 and Scmv2 were mapped to chromosomes 6S and 3L, respectively, by utilising QTL map** and bulked segregant analysis (BSA) [1, 12–14]. Additional three minor QTL were identified on chromosomes 1, 5, and 10 [1]. Presence of resistance alleles at both loci, Scmv1 and Scmv2, is crucial for complete SCMV resistance. Scmv1 suppresses symptoms at all developmental stages, Scmv2 at later stages of infection [1, 15]. One major MDMV resistance gene (Mdmv1) mapped to the same region of chromosome 6S as Scmv1. So far, it is not clear, whether or not Mdm1 and Scmv1 are the same or closely linked genes. The Scmv1/Mdmv1 chromosome region contains a cluster of resistance gene analogues [4, 1,
MDMV experiment: within-time- point analysis
Only two genes were significantly differentially expressed in the MDMV experiment at a FDR level of p ≤ 0.05 within time points. The two genes were significantly differentially expressed at two different time points and all were up-regulated in F7 SS/SS. One of the two genes (605018B04.x1) was also significantly differentially expressed within time points in the SCMV experiment. The fold of change did not exceed 3-fold for all three significant gene × time point combinations (data not shown).
GO description for 605018B04.x1 was binding activity (metallothionein-like protein), whereas no GO assignment but homology to a solanesyl diphosphate synthase was found for the second gene (947026D04.x1) http://www.tigr.org/.
MDMV experiment: between-time-point analysis
Forty-six percent out of 7260 observations showed significant differential expression between time points at a FDR p level ≤ 0.05. The majority of up-regulated differentially and significantly differentially expressed genes were found for T1 (see Additional file 3). Distribution of genes regarding their fold changes is shown in Additional file 4.
The two genes (605018B04.x1, 947026D04.x1) identified in the within-time-point analysis as significantly differentially expressed were also significantly differentially expressed in the between-time-point analysis (see Additional file 5).
SCMV experiment: quantitative RT-PCR
Six out of the 65 consistently differentially expressed sequences from microarray experiments were selected for validation by qRT-PCR based on their map position, expression pattern, fold of change in microarray experiments, or sequence homology related to resistance response genes. These included genes expressing a metallothionein-like protein, 26S ribosomal RNA, 14-3-3-like protein GF14-6, two genes for S-adenosylmethionine synthetase 1, and germin-like protein 4 (Table 3). An endogenous maize actin gene was used as a reference in this experiment. Coefficients of determination (R2) for reference and target genes were between 0.94 and 0.99, confirming good quality of standard curves. PCR efficiencies for target and reference genes ranged from 1.0 to 1.4, except of germin-like protein deviating from the standard PCR efficiency for target gene up to E = 3.6 (Table 4).
Differential expression of the metallothionein-like protein homologue (605018B04.x1) was validated by qRT-PCR with a fold change of 89.2 (average from four biological replications) as compared to 2.6 (p = 0.0) fold from microarray experiments. The S-adenosylmethionine synthetase 1 gene (946063C12.y1) and germin-like protein 4 (za72g09.b50) were validated with a fold of 2.7, as compared to 2.0 and 1.4 fold from microarrays, respectively. The putative 26S ribosomal RNA gene (605018B03.x1) and the S-adenosylmethionine synthetase 1 gene (946126A02.y1) were not validated when averaging four biological replications (1.6 and 1.2 fold, respectively), but had significant fold of changes in one of the four replications (data for separate replications not shown). The 14-3-3- like protein GF14-6 (Zm06_09h07_R) was not validated by qRT in any of the four biological replications. However, the fold value for three replications ranged from 1.6 to 1.7 fold.
Discussion
Validation and reliability of data
Comparison to previously published data
The purpose of this study was to identify and validate genes involved in resistance response to SCMV and MDMV. In previous SCMV experiments [18, 19], a set of candidate genes was identified to show significant differential expression between near-isogenic SCMV resistant and susceptible inbred lines. These genes, together with resistance gene analogs (RGAs) were spotted on our cDNA SCMV array. Twice as many genes based on earlier studies showed differential expression as compared to RGAs, indicating usefulness of pre-selection and reliability of microarray approach.
Comparison of SCMV and MDMV experiments
The MDMV experiment was set up to compare response of isogenic lines containing Scmv1 and/or Scmv2 regions from the resistant FAP1360A inbred line to related but different viruses. Comparative studies of related viruses displaying common symptoms in the same host offer an opportunity to link changes in global gene expression to specific symptoms and to identify common genes involved in resistance responses [22]. It was assumed in the experimental design that F7 RR/RR demonstrates full resistance to both SCMV and MDMV [31].
Additional comparative gene annotation was based on results obtained by Whitham et al. [21] on Arabidopsis infected with five distinct viruses, including a mosaic potyvirus. Three homologous genes (pathogenesis-related protein, alcohol dehydrogenase and glutathione S-transferase) were identified in the classes cell rescue, defence, death, and ageing of Arabidopsis, thus indicating the reliability of microarray technology for detection of pathogenesis-related genes.
Association of map positions of differentially expressed candidate genes with Scmv1 and Scmv2
A virus resistance gene needs to be expressed before pathogen invasion, in order to enable a rapid response after infection. Its expression may increase further after virus attack. In previous QTL experiments [1], Scmv1 (as QTL) was detectable at each scoring time point after inoculation, whereas Scmv2 became first detectable and induced at later scoring stages. Assuming that both Scmv1 and Scmv2 are single genes, clustering of differentially expressed genes in the Scmv1 and Scmv2 genomes regions could either be due to linkage drag of genes located in the polymorphic regions in isogenic line contrasts without effect on SCMV resistance (caused by heterozygosity), or clustering of genes involved in SCMV resistance in the Scmv1 and/or Scmv2 regions. Assuming that each of the two donor segments is 40 cM long, both regions would represent 5% of the total genome (80 cM out of 1600 cM average maize genome size). The percentage of candidate genes (one gene each) falling into either the Scmv1 (6.00–6.01) or Scmv2 bins (3.04–3.05) is 8% (2 out of 24 putative map locations for the 14 mapped differentially expressed genes, Table 2), which is not significantly different from the 0-hypothesis tested by the X2 test (no clustering of differentially expressed genes). Thus, no evidence of clustering of differentially expressed genes in the Scmv1 and Scmv2 regions was found, which also means, that differential gene expression of genes due to linkage drag was limited. Moreover, finding 2 out of 24 gene locations in agreement with the Scmv1 and Scmv2 genome locations is not significantly different from expectations based on probability theory. Thus, colocalization of differentially expressed genes is only a weak indicator for candidacy of being Scmv1 or Scmv2.
Time course data
In contrast to fungi and bacteria, viruses are directly transferred by specific vectors into host cells. The infection cycle includes virus disassembly, RNA translation and replication, new viral particle assembly, and movement. The time required for these processes may vary and be virus/host dependent.
Immediate response of plants against virus attack is obligatory for fast activation of defence mechanisms [32]. Significant differential expression of majority of genes in our study one hour post inoculation, drop** down to about one-third twelve hours post inoculation may be a result of such rapid responses of host plant to viral infection. The anticipation of detected genes in mechanical stress responses proved insignificant in applied mock control experiment.
To arque our statement we present the potyvirus study of Maule et al. [33], where induction of genes related to pathogenesis (putative protein targeting, virion assembly and trafficking) and to general stress responses was detected immediately after inoculation, similar to the study of Love et al. [34]. Furthermore, Marathe et al. [35], detected robust plant resistance responses at transcriptome level to potyvirus infection at early (at least three hours post inoculation) time points. Changes in gene expression due to responses initiated by specific interactions between virus and host proteins, for instance potyvirus coat protein VPg with plant eIF4 initiation factor have also been reported [36]. Moreover, rapid silencing and blockade of viral protein expression by modified RNAi was observed within one-two hours post inoculation [37]. In contrast, however Yang et al. [38] concluded that changes in gene expression depend on virus type and its accumulation (threshold of viral RNA and proteins) in infected tissues hence occur rather at later stages post inoculation. Similarly, Whitham et al. [39] stated that actual transcriptional changes depend on the progress of viral infection.
The conflicting observations of different research groups might be due to host-virus system specificity and need to be studied in more detail, before generalizations can be made.
Candidate genes and their involvement in signal transduction pathways
Metallothioneins are known to be involved in metal binding/metabolism and detoxification reactions in animals and yeasts [40, 41]. Slightly modified functions of metallothioneins have been reported in plants, where their increased expression was observed in senesced leaflet and abscission zones, under ethylene induction, or as a consequence of mechanical wounding when infecting tobacco with TMV virus was obsereved [42–44]. Moreover, a possible role of metallothioneins in controlling intracellular redox potential and activation of oxygen detoxification, a common strategy used by plants after pathogen invasion was suggested by Hamer [40]. Finding of metallothionein-like protein expressed in all time points (including mock control at significant fold of change) and for both viruses may suggest its differential expression as a cause of mechanical wounding. However, its participation in pathogen control cannot be ruled out.
S-adenosylmethionine synthetase is a key enzyme involved in generation of S-adenosylmethionine from methionine. S-adenosylmethionine is a major methyl donor in plants involved in polyamin and ethylene biosynthesis as well as in methylation reactions modifying lipids, proteins and nucleic acids [45–47]. Ethylene plays an important role in various plant disease resistance pathways. It has originally been considered a stress hormone due to its synthesis induced by stress signals, such as mechanical wounding, chemicals and metals, drought, extreme temperatures, and pathogen infection [46, 48]. Some pathogens can induce plant defence responses via activation of the ethylene signal transduction pathway, whereas plants deficient in ethylene signalling may show either increased susceptibility or increased resistance [49, 50]. Alternatively, methylation of the fully susceptible F7 SS/SS genotype might be reduced as revealed in our study by the upregulation of S-adenosylmethonine synthetase isoforms (946126A02.y1, 1091032B12.y1 a and b, 946063C12.y1) in all other genotypes. Resistance to SCMV and MDMV might depend on the methylation status of the plant, relating to post-transcriptional gene silencing mediated by HEN1 like methyltransferase [37].
Germins are water-soluble proteins expressed during seed germination in very young seedlings of wheat and barley. In mature leaves they are induced in response to pathogen attack [51]. In plants other than wheat and barley, sequences related to germins are termed "germin-like". Germins and germin-like proteins were isolated from hot pepper during resistance response to bacterial and viral infection [52, 53]. Pathogen response functions of germins were discovered with the identification of germin as an oxalate oxidase generating hydrogen peroxide. H2O2 is a catalyst of cell-wall reinforcement (oxidative cross-linking) and a basis for defence reactions in higher plants. The specific-pathogen-response OXO transcript was found in the wall of barley mesophyll cells six hours after inoculation with powdery mildew [54]. However, it is still unclear if germin-like proteins also have oxalate-oxidase activity and if their biological function is comparable to germins [55–59].
A common feature of expression patterns of genes in the resistant genotype F7 RR/RR is lack of signs of oxidative damage (downregulation of class III peroxidases and germins), whereas partially resistant and susceptible genotypes showed upregulation of genes controlling production of hydrogen peroxide. Furthermore, oxidative damage could affect chloroplasts, perturbing their proper function as shown for response to plum pox potyvirus [60]. The analysis of SCMV cylindrical inclusion (CI) virus protein (NP_734137), known to be involved in virus replication and cell-to-cell movement [61], with the ChloroP 1_1 CBS tool http://www.cbs.dtu.dk/services/ChloroP/ revealed a possible chloroplast transit peptide of 65 amino acids [62]. This could explain upregulation of the 14-3-3 proteins (Zm06_09h07_R) in genotypes other than the fully resistant genotype F7 RR/RR. 14-3-3 proteins are known to target the transit peptide to the chloroplast, where it will be cleaved upon entrance as shown for other potyvirus [79], and 3 exons from the eIF3E barley gene with duplication.
Hybridization design
144 and 24 arrays were utilised for the SCMV and the MDMV experiment, respectively. The SCMV experiment was carried out with all four near-isogenic genotypes. An unresolvable row-column design was optimized for six possible pairings of genotypes within each time point, where six rows corresponded to six slides and two columns corresponded to the two dyes. The MDMV experiment was carried out with two near isogenic genotypes: F7 SS/SS and F7 RR/RR, using a pair-wise dye-swap design.
Quantitative RT-PCR
Total RNA from leaf tissue (remaining sixteen harvested leaves per biological replication) of near isogenic genotypes was isolated using TRIzol reagent (Invitrogen GmbH, Karlsruhe, Germany). RNA purification was conducted on RNeasy mini kit columns (QiaGen AG, Hilden, Germany) following manufacturer's instructions, with previous DNA digestion with RNase free DNase (Qiagen AG, Hilden, Germany). RNA quality was checked on 1.2% formaldehyde agarose gels and quantification was done by spectrophotometry. Sequence-specific primers for real-time (RT) PCR were designed using Primer Express™ software, Version 1.5 (Applied Biosystems, Foster City, CA, USA) (Table 5).
QRT-PCR was conducted with One-Step QuantiTect SYBR® Green RT-PCR Kit (Qiagen AG, Hilden, Germany) on the ABI PRISM™ 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) under the following conditions: 50°C for 30 min, 95°C for 15 min and 45 cycles of 94°C for 30 sec, 58°C for 15 sec, and 72°C for 30 sec in total volumes of 25 μl reactions. Four biological and three technical replications were used for every gene in order to precisely quantify transcript abundance. Dissociation curve analyses were performed to identify primer-dimers and unspecific PCR products. An endogenous reference sequence was derived from the maize actin 1 gene (MAc1) [EMBL-EBI: J01238]
Statistics
Microarrays
Raw intensity and background values generated by Array Vision, version 8.0 (Imaging Research Inc., St. Catharines, Canada) were utilized for data analysis. The main interest was to determine the expression patterns of pair-wise contrasts between genotypes at the same time point (within-time-point analysis), whereas contrasts of a genotype at two different time points were of secondary interest (between-time-point analysis). Locally weighted scatterplot smoothing (LOWESS) regression was performed to adjust for differences within an array.
The following linear mixed model was fitted:
where y ijkl is the log2-signal intensity, g i fixed effect for genotype, t j fixed effect for the time point, a k random effect for the array, d l fixed effect for the dye, (g*t) ij genotype and time point interaction and (g*d) il genotype and dye interaction. The calculations were performed with the SAS System for Windows, Version 9.1.
Pair-wise contrasts between different genotype*time combinations in the SCMV experiment were estimated, considering only contrasts between genotypes within one time point and contrasts of one genotype at different time points. The corresponding FDR adjusted p-values and fold changes were determined. Least square means of genotype*time were calculated, i.e., the value of a certain genotype at a specific time point averaged over the other effects. The degrees of freedom for the tests were calculated according to the containment method. SAS (Institute Inc. (1999): SAS/STAT User's Guide, Version 8. Cary, NC). For MDMV data analysis the same linear model was fitted but separate variance terms for mock control and normal data were specified.
Blastn analysis in TIGR Unique Gene Indices http://www.tigr.org/plantgenomics/htdocs/blast_servers.html for maize was performed in order to reveal the putative function of unknown sequences from Arabidopsis thaliana, barley, maize, rice, rye and wheat, with a cut off e-value of 10 (Ros et al., 2004). Additional blastn analyses were performed in MIPS http://mips.gsf.de/ and IRGSP http://rgp.dna.affrc.go.jp/IRGSP/ databases for gaining maximum information about the genes of interest.
The calculations of significances for the number of genes between time points were calculated using the McNemar exact test (SAS System for Windows, Version 9.1).
QRT-PCR
Relative expression rates of the target genes were calculated as follows:
where E target is the PCR efficiency for the target gene and E ref is the PCR efficiency for the endogenous reference. PCR efficiencies (E = 10(-1/slope)-1), were derived from calibration data of serially diluted RNA: 100%, 50%, 10%, 5%, 1%, 0.5%, 0.1% and water. ΔCttarget and ΔCtref values were determined as described by Dilger et al. [80]. Baseline and threshold values were adjusted manually if necessary, as recommended by Applied Biosystems http://www.appliedbiosystems.com/support/tutorials/pdf/performing_rq_gene_exp_rtpcr.pdf.