A CC-NB-LRR gene confers leaf stripe resistance at the Rdg2a locus in barley

Abstract

Barley leaf stripe, caused by the seed-transmitted hemi-biotrophic fungus Pyrenophora graminea, is a barley disease particularly acute in Nordic countries, during spring sowing, and in the Mediterranean’s winter barley districts where causes severe yield losses. To date, only two P. graminea race-specific resistance genes are known: Rdg1a, identified in cultivar Vada, and Rdg2a, identified in cv. Thibaut. Rdg2a confers immunity to at least three different P. graminea monoconidian isolates, including the most widespread and virulent Italian isolate Dg2, but it is overcome by Dg5. The aim of the present work was to isolate this gene, characterize the Rdg2a locus and its evolution and mine the bases of Rdg2a-mediated resistance. In a previous analysis the map-based cloning and the sequencing of the Rdg2a locus were carried out. Three homolog R genes encoding CC-NB-LRR proteins, that represent the majior class of resistance proteins, were identified at the locus and were named as Nbs1-Rdg2a, Nbs2-Rdg2a and Nbs3-Rdg2a (Bulgarelli et al., 2010). To determine which of the three genes is the Rdg2a gene, we started the research investigating their structure. RACE analyses showed that for the third candidate (Nbs3-Rdg2a) alternative splicing processes determine the synthesis of severly truncated and probably non-functional proteins. This allowed us to excluded Nbs3- Rdg2a from acting in resistance to isolate Dg2. By means RT-PCRs on a pair of Near Isogenic Lines (NIL) that differ only for alleles at the Rdg2a locus (susceptible cv. Mirco and its resistant NIL, NIL3876), we demonstrated that both Nbs1-Rdg2a and Nbs2-Rdg2a are transcribed in embryos (where the resistance takes place) and leaf tissues of the resistant line, but they are not expressed in the susceptible nearisogenic phenotype. Sequencing of the Mirco alleles revealed rearrangements in the putative promoter regions: two insertions, one next to a putative TATA-box element and the other, carrying terminal inverted repeats, in the 5’ UTR, for Nbs1-rdg2a, and a deletion just at the level of a MITE-like element, present in Thibaut Nbs2-Rdg2a, for Nbs2-rdg2a. It is likely that these changes represent the cause of the lacking of expression of these genes in the susceptible genotype. Moreover, qRT-PCRs showed that Nbs1-Rdg2a transcription was un-responsive to P. graminea infection, while Nbs2-Rdg2a transcripts increased during the first stages of infection. However, Nbs2-Rdg2a mRNA abundance was significantly lower than that of Nbs1-Rdg2a. To define which gene is the Rdg2a gene, a complementation assay was conducted. Susceptible cv. Golden Promise was transformed by agoinfiltration with each gene independently, under the control of its native promoter. Interestingly, the rescue of Dg2-resistance was observed only when plants were transformed with Nbs1-Rdg2a (90-100% of resistance), suggesting that this gene is Rdg2a. Comparing the sequences of the three genes at the Rdg2a locus, we found that similarly to other R genes, Rdg2a underwent to diversifying selection, according to a model in which resistance genes co-evolves with pathogen effector(s) gene(s). The fact that the Rdg2a locus contains a gene cluster of highly similar sequences has most likely contributed to significative rearrangements during evolution, probably derived from unequal crossovers resulting in sequence exchange between paralogs and, possibly, in the generation of recombinant genes, as well as to expansion/contraction of gene copy number. Regarding this last case, we have also characterized the rdg2a locus of the susceptible cv. Morex. Morex rdg2a locus carries two deletions and the rdg2a allele might derived from an un-equal crossingover between Rdg2a and Nbs2-Rdg2a ancestors that led to a reduction of the number of the gene family members. Most resistance proteins function through inducing a Programmed Cell Death (PCD) that lead to a Hypersensitive Response (HR) at the level of the infected cells. Histological analyses using the TUNEL method did not reveal any significative difference in PCD between infected embryos of resistant and susceptible varieties and the number of cells undergoing PCD was transcurable. These finding let us to conclude that the Rdg2a-mediated resistance does not involve HR but it is most likely based on the strengthening of physical and chemical barriers in the cell walls and intercellular spaces of the embryo tissues. In conclusion, we identified, cloned and characterized the first resistance gene active against a seed-borne disease; importantly, the gene belongs to the poorly represented class of R genes which does not trigger a hypersensitive responce.