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.
