Genetic Diversity and Virulence of Wheat and Barley Strains of Xanthomonas translucens from the Upper Midwestern United States.

Bacterial leaf streak (BLS) of wheat and barley, caused by Xanthomonas translucens pv. undulosa and X. translucens pv. translucens, has been of growing concern in small grains production in the Upper Midwestern United States. To optimize disease resistance breeding, a greater awareness is needed of the pathovars and genetic diversity within the pathogens causing BLS in the region. Multilocus sequencing typing (MLST) and analysis (MLSA) of four common housekeeping genes (rpoD, dnaK, fyuA, and gyrB) was used to evaluate the genetic diversity of 82 strains of X. translucens isolated between 2006 and 2013 from wheat, barley, rye, and intermediate wheatgrass. In addition, in planta disease assays were conducted on 75 strains to measure relative virulence in wheat and barley. All strains were determined by MLSA to be related to X. translucens pv. undulosa and X. translucens pv. translucens. Clustering of strains based on Bayesian, network, and minimum spanning trees correlated with relative virulence levels in inoculated wheat and barley. Thus, phylogeny based on rpoD, dnaK, fyuA, and gyrB correlated with host of isolation and was an effective means for predicting virulence of strains belonging to X. translucens pv. translucens and X. translucens pv. undulosa.

Phi-class glutathione-S-transferase is involved in Dn1-mediated resistance.

Wheat Dn genes afford resistance to the economically important pest, Diuraphis noxia (Kurdjumov, Russian wheat aphid, RWA) and have been the topic of transcriptomic and proteomic studies aimed at unraveling the pathways involved in resistance. The antibiotic resistance conveyed by Dn1 is characterized by a hypersensitive response (HR) followed by systemic acquired resistance (SAR). Although many candidate genes differentially expressed during the Dn1-mediated resistance response have been identified, few have been functionally verified. The aim of this study was to silence three HR-associated candidate genes in Dn1 containing wheat using virus-induced gene silencing (VIGS): thylakoid-associated ascorbate peroxidase (tAPX), phi-class glutathione-S-transferase (TaGSTF6) and superoxide dismutase Cu/Zn (SOD). D. noxia fertility was used as a measure of antibiotic resistance. Silencing of SOD Cu/Zn had little effect on D. noxia fertility, while increased aphid reproduction was recorded on tAPX- and TaGSTF6-silenced plants. However, tAPX-silencing only affected early measurements and did not have a prolonged effect on resistance. TaGSTF6-silenced plants expressed lowered H2 O2 production in resistant wheat under infestation conditions, suggesting that TaGSTF6 and H2 O2 play an integral role in Dn1-mediated D. noxia resistance in wheat plants.

Near-isogenic lines of Triticum aestivum with distinct modes of resistance exhibit dissimilar transcriptional regulation during Diuraphis noxia feeding.

Russian wheat aphid (Diuraphis noxia, Kurdjumov) feeding on susceptible Triticum aestivum L. leads to leaf rolling, chlorosis and plant death - symptoms not present in resistant lines. Although the effects of several D. noxia (Dn) resistance genes are known, none have been isolated or characterized. Wheat varieties expressing different Dn genes exhibit distinct modes of D. noxia resistance, such as antibiosis (Dn1), tolerance (Dn2), and antixenosis (Dn5). However, the mechanism whereby feeding aphids are perceived, and how subsequent transcriptional responses are partitioned into resistance categories, remains unclear. Here we report on downstream events in near-isogenic wheat lines containing different Dn genes after D. noxia biotype SA1 feeding. Transcripts involved in stress, signal transduction, photosynthesis, metabolism and gene regulation were differentially regulated during D. noxia feeding. Expression analyses using RT-qPCR and RNA hybridization, as well as enzyme activity profiling, provide evidence that the timing and intensity of pathways induced are critical in the development of particular modes of resistance. Pathways involved include the generation of kinase signalling cascades that lead to a sustained oxidative burst, and a hypersensitive response that is active during antibiosis. Tolerance is a passive resistance mechanism that acts through repair or de novo synthesis of photosystem proteins. Results further suggest that ethylene-mediated pathways are possibly involved in generating volatile compounds and cell wall fortification during the antixenosic response.

Virus-induced gene silencing suggests (1,3;1,4)-ß-glucanase is a susceptibility factor in the compatible russian wheat aphid-wheat interaction.

The Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), is a significant insect pest of wheat (Triticum aestivum L.) and has a major economic impact worldwide, especially on winter wheat in the western United States. The continuing emergence of new RWA biotypes virulent to existing resistance genes reinforces the need for more durable resistance. Studies have indicated that resistance in previously susceptible plants can be produced by knock-down of susceptibility genes or other genes involved in host plant susceptibility. Therefore, investigation into genes involved in compatible RWA-wheat interactions could be a feasible approach to achieving durable RWA resistance. The objective of this study was to test whether silencing (1,3;1,4)-ß-glucanase, previously observed to be highly induced in susceptible compared with resistant wheat during aphid infestation, would confer resistance to a susceptible wheat genotype. Barley stripe mosaic virus-mediated virus-induced gene silencing was employed to test whether (1,3;1,4)-ß-glucanase is involved in the susceptible reaction of 'Gamtoos-S' (GS). Controlled infestation with U.S. biotype RWA2 was done to assess aphid reproduction and host symptom development. Aphids on (1,3;1,4)-ß-glucanase-silenced plants reproduced less per day and had longer prenymphipositional periods than those on control GS plants. Furthermore, the (1,3;1,4)-ß-glucanase-silenced plants exhibited less chlorosis and greater dry weight compared with GS. Aphid reproduction and host plant symptom development showed linear relationships with (1,3;1,4)-ß-glucanase transcript levels. Our results suggest that (1,3;1,4)-ß-glucanase is required for successful infestation by the RWA and may be a susceptibility factor that could be exploited as a potential target for RWA resistance breeding.

Hypervirulent Diuraphis noxia (Hemiptera: Aphididae) biotype SAM avoids triggering defenses in its host (Triticum aestivum) (Poales: Poaceae) during feeding.

In the molecular arms race between aphids and plants, both organisms rely on adaptive strategies to outcompete their evolutionary rival. In the current study, we investigated the difference in elicited defense responses of wheat (Triticum aestivum L.) near-isogenic lines with different Dn resistance genes, upon feeding by an avirulent and hypervirulent Diuraphis noxia Kurdjumov biotype. After measuring the activity of a suite of enzymes associated with plant defense, it became apparent that the host does not recognize the invasion by the hypervirulent aphid because none of these were induced, while feeding by the avirulent biotype did result in induction of enzyme activity. Genomic plasticity in D. noxia may be a likely explanation for the observed differences in virulence between D. noxia biotype SA1 and SAM, as demonstrated in the current study.

The transcriptional network of WRKY53 in cereals links oxidative responses to biotic and abiotic stress inputs.

The transcription factor WRKY53 is expressed during biotic and abiotic stress responses in cereals, but little is currently known about its regulation, structure and downstream targets. We sequenced the wheat ortholog TaWRKY53 and its promoter region, which revealed extensive similarity in gene architecture and cis-acting regulatory elements to the rice ortholog OsWRKY53, including the presence of stress-responsive abscisic acid-responsive elements (ABRE) motifs and GCC-boxes. Four proteins interacted with the WRKY53 promoter in yeast one-hybrid assays, suggesting that this gene can receive inputs from diverse stress-related pathways such as calcium signalling and senescence, and environmental cues such as drought and ultraviolet radiation. The Ser/Thr receptor kinase ORK10/LRK10 and the apoplastic peroxidase POC1 are two downstream targets for regulation by the WRKY53 transcription factor, predicted based on the presence of W-box motifs in their promoters and coregulation with WRKY53, and verified by electrophoretic mobility shift assay (EMSA). Both ORK10/LRK10 and POC1 are upregulated during cereal responses to pathogens and aphids and important components of the oxidative burst during the hypersensitive response. Taken with our yeast two-hybrid assay which identified a strong protein-protein interaction between microsomal glutathione S-transferase 3 and WRKY53, this implies that the WRKY53 transcriptional network regulates oxidative responses to a wide array of stresses.

Virus-induced gene silencing of WRKY53 and an inducible phenylalanine ammonia-lyase in wheat reduces aphid resistance.

Although several wheat genes differentially expressed during the Russian wheat aphid resistance response have recently been identified, their requirement for and specific role in resistance remain unclear. Progress in wheat-aphid interaction research is hampered by inadequate collections of mutant germplasm and difficulty in transforming hexaploid wheat. Virus-induced gene silencing (VIGS) technology is emerging as a viable reverse genetics approach in cereal crops. However, the potential of VIGS for determining aphid defence gene function in wheat has not been evaluated. We report on the use of recombinant barley stripe mosaic virus (BSMV) to target and silence a WRKY53 transcription factor and an inducible phenylalanine ammonia-lyase (PAL) gene, both predicted to contribute to aphid defence in a genetically resistant wheat line. After inoculating resistant wheat with the VIGS constructs, transcript abundance was reduced to levels similar to that observed in susceptible wheat. Notably, the level of PAL expression was also suppressed by the WKRY53 construct, suggesting that these genes operate in the same defence response network. Both knockdowns exhibited a susceptible phenotype upon aphid infestation, and aphids feeding on silenced plants exhibited a significant increase in fitness compared to aphids feeding on control plants. Altered plant phenotype and changes in aphid behaviour after silencing imply that WKRY53 and PAL play key roles in generating a successful resistance response. This study is the first report on the successful use of VIGS to investigate genes involved in wheat-insect interactions.