Molecular Characterization of Erysiphe necator (powdery mildew) resistance loci from the grapevine cultivar `Regent´ and their signaling network
Final Report Abstract
This project delimited the Erysiphe necator (powdery mildew, PM) resistance locus Ren9 and differentiated it clearly from the previously elaborated locus Ren3. Both loci reside on chromosome 15 in the resistant grapevine cultivar ‘Regent’. Using recombinants it was shown that they act independently. Ren3 and Ren9 both mediate a post-penetration inhibition of the pathogen caused by hypersensitive responses, such as production of reactive oxygen species. Resistance mediated by Ren9 seems to be more efficient than Ren3 under field conditions. New genetic markers (Indels) were designed to follow the Ren9 locus closely in marker-assisted selection approaches in grapevine breeding. These have been published and were directly transferred to the breeding department of the Institute for application. The genome of resistant ‘Regent’ was sequenced in cooperation with Pablo Carbonell and Detlef Weigel at MPI Tübingen to very high quality level. Using the Trio Canu approach, the two haplophases making up the ‘Regent’ genome, the resistance-carrying haplophase from ‘Chambourcin’, and the susceptible V. vinifera haplophase inherited from ‘Diana,’ were separated. Genes were predicted and annotated to 92% completeness. Isoform-level transcriptome sequencing was performed on ‘Regent’ samples from comprehensive sampling of various plant tissues and PM inoculated samples collected at two time points after pathogen application (12 and 24 hpi). This dataset will be helpful to not only study resistance genes, but also for the analysis of tissue specific expression of transcripts and their possible isoform splice variants. This data set was applied to study transcriptional variants in the Ren3 and Ren9 candidate gene region. The NLR-type resistance genes RGA2 and RGA3 in the Ren3 locus are only transcribed form the resistance carrying haplophase. Upon pathogen challenge, RGA2 produces a transcript with all functional NLR domains (CC, NB-Arc, LRR), while it is transcribed to a truncated version in flowers. In several tissues, a combined transcript with inverse orientation of RGA2 and RGA3 was observed, that can form a double stranded RNA hairpin, which is known for the initiation of RNA interference. These observations suggest a gene regulation on RNA level to be further elucidated. The finding of a small RNA targeting a different NLR gene on chromosome 12 in small RNA seq studies supports this conclusion. The RLPK (receptor like protein kinase) gene in locus Ren9 is exclusively expressed in senescent leaves in the susceptibility-linked haplophase of ‘Diana’, while its transcription is observable for both PM inoculated samples (12hpi and 24hpi), ‘Leaf’, ‘Shoot’ and also the ‘Senescent Leaf’ in the resistance carrying haplotype of ‘Chambourcin’. This provides evidence that this gene is involved in the resistance response towards powdery mildew. Further functional characterization is necessary. Additional RNASeq (mRNA and small RNA) analysis after inoculation of ‘Regent’ with powdery mildew indicated upregulation of specific sets of phenylalanine-ammoniumlyase-(PAL-) and stilbene synthase (STS-) genes in the case of resistance. Stilbenes are the major phytoalexins of grapevine and synthesized by a large family of STS genes from one branch of the phenylpropanoid metabolism, which has PAL activity as entry point. Furthermore, receptor-like kinase genes and glutathione reductase genes in addition to multicopper oxidases possibly involved in ROS production were upregulated. Altogether, this research provided many new tools to study the molecular physiology of grapevine. It delimited resistance gene candidates in the loci Ren3 and Ren9 and gave deep insights into their modulation in the case of early powdery mildew challenge.
Publications
- (2017): Fine mapping of Ren3 reveals two loci mediating hypersensitive response against Erysiphe necator in grapevine. Euphytica (2017) 213:68
Zendler D, Schneider P, Töpfer R and Zyprian E
(See online at https://doi.org/10.1007/s10681-017-1857-9) - (2017): Powdery mildew responsive genes of resistant grapevine cultivar ‚Regent‘. Vitis 56, 181-188 (2017)
Welter LJ, Tisch C, Kortekamp A, Töpfer R and Zyprian E
(See online at https://doi.org/10.5073/vitis.2017.56.181-188) - (2018) The Ren3 locus of ‘Regent’ co-segregates with a NBS-LRR gene cluster conferring resistance to powdery mildew by hypersensitive response Poster discussion at XII International Conference on Grapevine Breeding and Genetics, 2018, Bordeaux, France
Zendler D, Schneider P, Töpfer R and Zyprian E
- (2019): Genetic rearrangements in the resistance locus Ren3 lead to disruption of a CC-NBS-LRR gene in susceptible grapevines. Oral presentation at Gesellschaft für Pflanzenzüchtung - AG Genomanalyse, Meeting 2019, University of Hohenheim, Germany
Zendler D, Töpfer R and Zyprian E
- (2020): Confirmation and fine mapping of the resistance locus Ren9 from the grapevine cultivar ‘Regent’. BioRxiv preprint server
Zendler D, Töpfer R and Zyprian E
(See online at https://doi.org/10.1101/2020.11.27.400770) - (2021): Confirmation and Fine Mapping of the Resistance Locus Ren9 from the Grapevine Cultivar ‘Regent’. Plants 2021, 10, 24
Zendler D, Töpfer R, Zyprian E
(See online at https://doi.org/10.3390/plants10010024)
