Characterization of a foxtail mosaic virus vector for gene silencing and analysis of innate immune responses in Sorghum bicolor
Melissa BredowMartha Ibore NatukundaBliss M. BeerninkAline Sartor ChicowskiMaria G. Salas FernandezSteven A. Whitham
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Abstract:
Sorghum is vulnerable to many biotic and abiotic stresses, which cause considerable yield losses globally. Efforts to genetically characterize beneficial sorghum traits, including disease resistance, plant architecture, and tolerance to abiotic stresses, are ongoing. One challenge faced by sorghum researchers is its recalcitrance to transformation, which has slowed gene validation efforts and utilization for cultivar development. Here, we characterize the use of a foxtail mosaic virus (FoMV) vector for virus-induced gene silencing (VIGS) by targeting two previously tested marker genes: phytoene desaturase (PDS) and ubiquitin (Ub). We additionally demonstrate VIGS of a subgroup of receptor-like cytoplasmic kinases (RLCKs) and report the role of these genes as positive regulators of early defence signalling. Silencing of subgroup 8 RLCKs also resulted in higher susceptibility to the bacterial pathogens Pseudomonas syringae pv. syringae (B728a) and Xanthomonas vasicola pv. holcicola, demonstrating the role of these genes in host defence against bacterial pathogens. Together, this work highlights the utility of FoMV-induced gene silencing in the characterization of genes mediating defence responses in sorghum. Moreover, FoMV was able to systemically infect six diverse sorghum genotypes with high efficiency at optimal temperatures for sorghum growth and therefore could be extrapolated to study additional traits of economic importance.Keywords:
Phytoene desaturase
Pseudomonas syringae
Sorghum is vulnerable to many biotic and abiotic stresses, which cause considerable yield losses globally. Efforts to genetically characterize beneficial sorghum traits, including disease resistance, plant architecture, and tolerance to abiotic stresses, are ongoing. One challenge faced by sorghum researchers is its recalcitrance to transformation, which has slowed gene validation efforts and utilization for cultivar development. Here, we characterize the use of a foxtail mosaic virus (FoMV) vector for virus-induced gene silencing (VIGS) by targeting two previously tested marker genes: phytoene desaturase (PDS) and ubiquitin (Ub). We additionally demonstrate VIGS of a subgroup of receptor-like cytoplasmic kinases (RLCKs) and report the role of these genes as positive regulators of early defence signalling. Silencing of subgroup 8 RLCKs also resulted in higher susceptibility to the bacterial pathogens Pseudomonas syringae pv. syringae (B728a) and Xanthomonas vasicola pv. holcicola, demonstrating the role of these genes in host defence against bacterial pathogens. Together, this work highlights the utility of FoMV-induced gene silencing in the characterization of genes mediating defence responses in sorghum. Moreover, FoMV was able to systemically infect six diverse sorghum genotypes with high efficiency at optimal temperatures for sorghum growth and therefore could be extrapolated to study additional traits of economic importance.
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RPG1 and RPM1 are disease resistance genes in soybean and Arabidopsis, respectively, that confer resistance to Pseudomonas syringae strains expressing the avirulence gene avrB. RPM1 has recently been demonstrated to have a second specificity, also conferring resistance to P. syringae strains expressing avrRpm1. Here we show that alleles, or closely linked genes, exist at the RPG1 locus in soybean that are specific for either avrB or avrRpm1 and thus can distinguish between these two avirulence genes.
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Abstract Genome-wide motif searches identified 1134 genes in the lettuce reference genome of cv. Salinas that are potentially involved in pathogen recognition, of which 385 were predicted to encode nucleotide binding-leucine rich repeat receptor (NLR) proteins. Using a maximum-likelihood approach, we grouped the NLRs into 25 multigene families and 17 singletons. Forty-one percent of these NLR-encoding genes belong to three families, the largest being RGC16 with 62 genes in cv. Salinas. The majority of NLR-encoding genes are located in five major resistance clusters (MRCs) on chromosomes 1, 2, 3, 4, and 8 and cosegregate with multiple disease resistance phenotypes. Most MRCs contain primarily members of a single NLR gene family but a few are more complex. MRC2 spans 73 Mb and contains 61 NLRs of six different gene families that cosegregate with nine disease resistance phenotypes. MRC3, which is 25 Mb, contains 22 RGC21 genes and colocates with Dm13. A library of 33 transgenic RNA interference tester stocks was generated for functional analysis of NLR-encoding genes that cosegregated with disease resistance phenotypes in each of the MRCs. Members of four NLR-encoding families, RGC1, RGC2, RGC21, and RGC12 were shown to be required for 16 disease resistance phenotypes in lettuce. The general composition of MRCs is conserved across different genotypes; however, the specific repertoire of NLR-encoding genes varied particularly of the rapidly evolving Type I genes. These tester stocks are valuable resources for future analyses of additional resistance phenotypes.
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SKIP, a component of the spliceosome, is involved in numerous signaling pathways. However, there is no direct genetic evidence supporting the function of SKIP in defense responses. In this paper, two SKIPs , namely, SlSKIP1a and SlSKIP1b , were analyzed in tomato. qRT-PCR analysis showed that the SlSKIP1b expression was triggered via Pseudomonas syringae pv. tomato ( Pst ) DC3000 and Botrytis cinerea ( B. cinerea ), together with the defense-associated signals. In addition, the functions of SlSKIP1a and SlSKIP1b in disease resistance were analyzed in tomato through the virus-induced gene silencing (VIGS) technique. VIGS-mediated SlSKIP1b silencing led to increased accumulation of reactive oxygen species (ROS), along with the decreased expression of defense-related genes (DRGs) after pathogen infection, suggesting that it reduced B. cinerea and Pst DC3000 resistance. There was no significant difference in B. cinerea and Pst DC3000 resistance in TRV- SlSKIP1a -infiltrated plants compared with the TRV- GUS -silencing counterparts. As suggested by the above findings, SlSKIP1b plays a vital role in disease resistance against pathogens possibly by regulating the accumulation of ROS as well as the expression of DRGs.
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The molecular nature of many plant disease resistance ( R ) genes is known; the largest class encodes nucleotide-binding site-leucine-rich repeat (NBS-LRR) proteins that are structurally related to proteins involved in innate immunity in animals. Few genes conferring disease susceptibility, on the other hand, have been identified. Recent identification of susceptibility to the fungus Cochliobolus victoriae in Arabidopsis thaliana has enabled our cloning of LOV1 , a disease susceptibility gene that, paradoxically, is a member of the NBS-LRR resistance gene family. We found LOV1 mediates responses associated with defense, but mutations in known defense response pathways do not prevent susceptibility to C. victoriae . These findings demonstrate that NBS-LRR genes can condition disease susceptibility and resistance and may have implications for R gene deployment.
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