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Discovering susceptibility genes to Rhizoctonia solani in rice as breeding targets for sheath blight disease resistance

Periodic Reporting for period 1 - RiZeSisT (Discovering susceptibility genes to Rhizoctonia solani in rice as breeding targets for sheath blight disease resistance)

Periodo di rendicontazione: 2018-09-01 al 2020-08-31

Conclusion of the action:
In “RiZeSisT” to breed for broad-spectrum resistance in rice to the sheath blight disease (ShBD), caused by the soil-borne fungus Rhizoctonia solani AG1-IA, we proposed to employ altered susceptibility genes (S genes). The molecular mechanisms underlying susceptibility to R. solani are largely unknown. Therefore, throughout this MSCA-IF, these mechanisms were dissected by using an -omics tool box. 21 candidate S genes were identified and the majority where functionally validated for their response to R. solani. The most promising genes may become the next targets to generate rice varieties with improved resistance to ShBD.

Summary:
Rice (Oryza sativa L.) is the second most grown cereal worldwide. However, its production is largely affected by the sheath blight disease (ShBD) caused by the soil-borne fungus Rhizoctonia solani Kühn, AG1-IA. To date, some level of partial resistance in rice has been identified, expressed by a number of QTLs, but germplasm with complete resistance is not available. This pathogen survives by forming sclerotia or dormant mycelia, infects rice during flooding and causes necrotic lesions on leaf sheaths and blades that lead to a strong yield reduction. Recently, the incidence and severity of ShBD in rice has increased due to the large-scale deployment of semi-dwarf varieties, high plant densities and the intensified application of nitrogen fertilizers. To date, control of ShBD is achieved by extensive use of fungicides. Therefore, it is essential to develop resistant varieties as an alternative to the use of chemical control. To breed for broad-spectrum resistance in rice we proposed to employ altered susceptibility genes (S genes). A S gene describes any plant gene that makes them vulnerable to infection by supporting a compatible interaction with the pathogen enabling it to grow and infect. The ultimate goal is to disable susceptibility gene function to limit the interaction with the attacker and disrupt the ability of the pathogen to induce disease. The molecular mechanisms underlying susceptibility to R. solani are largely unknown and therefore, “RiZeSisT” -Rice RhiZoctonia reSisTance-, aimed to identify essential rice candidate S genes required for R. solani infection (discovery) and to test whether mutations in these genes provide resistance to sheath blight disease (functional validation). The selected genes will become the next targets to generate rice varieties with improved resistance to ShBD.
The molecular mechanisms underlying susceptibility to R. solani are largely unknown. Therefore, in RiZeSisT we tried to dissect these mechanisms by using an -omics tool box approach: 1) transcriptomics, an (adapted) reproducible micro-chamber method was used for an infection time course experiment (focused on the presumed biotrophic phase of the pathogen) for RNA-seq with a susceptible rice variety and a R. solani AG1 IA strain. Using the proprietary technology platform from KeyGene, KeySeeQ®, an efficient analysis of the transcriptome data for lead discovery and ranking of high quality candidate S genes was performed. From 1226 differentially expressed genes, 135 and 1091 were down- or up-regulated, respectively; 2) effectoromics, we aimed to find pathogen-derived, conserved and early expressed effectors and their host targets. Using Oxford Nanopore Technologies®, PromethION, five strains of R. solani AG1 IA (isolated from infected rice) from Japan, India and Arkansas were sequenced and annotated. The five genomes were used to perform a candidate effector prediction analysis using a proprietary pipeline from KeyGene. In total more than 336 clusters were discovered of which some were recently described in the literature. After these approaches, 21 candidate S genes were selected for further investigation. Due to the turnaround time and the COVID-19 pandemic, the most promising candidate S genes (and some close orthologues) were selected as targets to generate mutants in collaboration with the University of Milan (Laboratory of Prof. Martin Kater). The lab of Prof. Kater was also host of the fellow to perform her Secondment to learn and have hands-on experience with novel approaches for gene-editing. A detached leaf assay was adapted and improved to screen T2 plants of the candidate S genes mutants to determine their response to R. solani. Furthermore, the fellow together with experts of KeyGene developed a computer vision algorithm to estimate the leaf lesion length and area. With this approach, the majority of candidate S genes where evaluated for their response to R. solani.

The fellow had ample opportunities to disseminate and communicate her research and findings. She presented her data in a poster in one national and two international conferences, 12 internal presentations and two external company visits; she was invited as speaker/panelist at two international and one national events at University (1) and primary (1) and secondary (1) school level; she co-organized a (one-day) scientific symposium and an innovation day at KeyGene; she published two non-scientific articles and more than four research pieces for the internal newsletter of the company. In addition, until now, one scientific publication is in preparation to be submitted. Although the fellow did not have an own project website, she was very active in social media, namely LinkedIn and Twitter (own profile and company profile), hashtags: #RiZeSisT #rice #Rhizoctonia #sheathblight #H2020 #MSCA #arroz, with some posts with more than 3000 views.
In RiZeSisT, candidate S genes were identified for the interaction between rice and R. solani which have not been reported before and represent a new strategy to provide broad-spectrum and durable resistance to ShBD. The impact of these results can be reflected in breeding programs (interested companies) generating improved rice varieties that resist damage by R. solani, and if translation is possible, of other crops as well. Some of the candidate S genes are conserved and have orthologues in other economically relevant crops attacked by this pathogen. Generating healthy rice plants will aid in meeting the global agriculture and food production goals to feed our growing world population which is paramount to safeguard economic stability and the citizens’ well-being.

The fellow reached end users by publishing a summary of RiZeSisT in the KeyNews (more than 200 on-line subscribers, including shareholders and breeders) and the LinkedIn company profile (more than 6,500 followers). In order to make policy makers and end-users aware of the potential of the project, two non-scientific articles were published: 1) in the EU_CORDIS, Plant Health Pack, 2021. This new edition showcases 12 EU-funded projects at the forefront of research and innovation activities in this domain and 2) the Annual Prophyta, 2021. This journal appears yearly at the occasion of the ISF World Seed Congress and is aimed at opinion leaders, researchers and companies in the seed industry.
A summary of RiZeSisT