Phenotypic analysis of magnaporthe oryzae mutants: a view into components of mRNM decay

Abstract

Magnaporthe oryzae is a filamentous fungus responsible for causing rice blast disease. Given this disease can cause yield losses of 90% during epidemic seasons, the importance of studying this organism’s pathogenicity is critical to farmers as the need for rice has increased for an exponentially growing global population. Whole genome studies in this organism have revealed genes with putative roles in virulence. The specific focus of my research is to examine gene regulation by mRNA degradation as one such virulence control mechanism through the analysis of XRN1 and CAF16, components in mRNA degradation. The exoribonuclease, XRN1, moves in a 5’ to 3’ manner to digest target mRNAs while CAF16 is a subunit within the CCR4-NOT1 complex that deadenylates the target mRNA. We generated targeted deletion mutants of XRN1 (Δxrn1) and of CAF16 (Δcaf16) for use in various assays to determine their phenotypic response. Mutants were grown in several stressors (i.e. ROS, nitrogen starved, osmotic stress) as well as non-stressors (i.e. complete media) and analyzed through growth rates. Results show that none of these stressors affect the mutants' ability to grow, indicating that these genes are not critical for growing under stressful conditions. Molecular profiling of the mutants under these stress conditions revealed inconsistent results regarding expression of XRN1 in ΔCAF16 and vice versa. Expression of two additional genes involved in mRNA degradation pathways, DCS1 and POR1, were measured to determine if there was a decrease in expression in Δ xrn1. In the absence of XRN1, the data revealed an increase in expression in both genes thereby suggesting both POR1 and DCS1 are involved in mRNA degradation, as previous studies in yeast suggested. Finally, mutants were inoculated onto a M. oryzae susceptible cultivar of barley to determine phenotypic differences. Data suggests reduced virulence in Δxrn1 as there were lesser, smaller lesions on the inoculated plant leaves. This phenotypic analysis suggests that XRN1, a component in mRNA degradation, is related to virulence, and likely works together with DCS1 and POR1 to mediate mRNA degradation. How mRNA degradation and virulence in this pathogen are linked, are fascinating subjects for a future study.