Vergleich der Wirt- und Nichtwirt-Interaktion von Gerste mit verschiedenen Magnaporthe-Arten anhand von histologischen Untersuchungen, Transkriptomstudien und funktionalen Genanalysen

  • Comparison of the host and nonhost interaction of barley with different Magnaporthe species by histological investigations, transcriptomics and functional gene analyses

Delventhal, Rhoda; Schaffrath, Ulrich (Thesis advisor); Slusarenko, Alan (Thesis advisor)

Aachen (2015, 2016)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen, 2015

Abstract

Wheat and barley crops are essential for world food security. Both crop species are susceptible to Magnaporthe oryzae, the causal agent of rice blast. However, both plant species are completely and stably resistant against isolates of a Magnaporthe species pathogenic on the grass genus Pennisetum. The mechanisms of this ‘nonhost resistance‘ are not yet fully understood, and the present thesis aimed at identifying genes contributing to its establishment.Thus, microarray analyses of the barley transcriptome during a host interaction (HI) with a M. oryzae isolate and during a nonhost interaction (NHI) with a Pennisetum-associated Magnaporthe isolate were performed. The role of individual candidate genes was investigated in more detail by relating their expression patterns to cellular infection stages observed by microscopy and by the targeted reduction of transcript abundance using a virus-induced gene silencing (VIGS) approach.In both HI and NHI the barley transcriptome responded to Magnaporthe inoculation with an altered, mostly up-regulated, expression of several thousand genes. Among the genes underlying this general transcriptional re-programming 189 candidate genes were identified whose expression was higher during NHI than during HI at aquivalent timepoints, and which could therefore play a role in nonhost resistance. In VIGS experiments the reduced expression of some single candidate genes did not cause a complete break of nonhost resistance, but did affect plant defense reactions in a quantitative manner. The role of the previously identified candidate gene CYP96B22 was analysed in more detail. This gene encodes a putative cytochrome P450 monooxygenase and its expression was induced by 6 h p.i. in NHI. In fact, the pre-penetration growth of both adapted and non-adapted Magnaporthe isolates on the leaf surface already triggered an enhanced transcript accumulation of CYP96B22. This was evidenced, when appressorium formation was reduced or absent, either after fungicide treatment or in a fungal mutant, and CYP96B22 induction still occurred. Therefore, it was concluded that barley and Magnaporthe isolates exchange signals across the intact plant surface before penetration. Microscopy revealed a decrease in effective papillae in the NHI and an increase of invasive pathogen growth in the HI after reduction of CYP96B22 transcript abundance by VIGS. This indicates a crucial function of CYP96B22 in the penetration resistance against Magnaporthe. Since other members of the CYP96 family have been shown to be involved in the biosynthesis of epicuticular waxes, CYP96B22 may contribute to the provision of wax components for structural barriers or to the synthesis of signal molecules.The barley transcriptome analysis was part of the ERA-PG project "TritNONHOST", in which HI and NHI of barley and wheat with three different pathogens (Magnaporthe, Blumeria and Puccinia) were investigated using a similar experimental setup. Data analyses revealed a large, overlapping gene set underlying the general transcriptional reprogramming in all pathogen interactions. However, nonhost resistance candidate genes identified for Blumeria or Puccinia interactions were different from those identified for the Magnaporthe interaction. These results account for a specific transcriptional nonhost response depending on the pathogen, and are in agreement with results for barley published previously by our group. In a project focusing on the fungal contribution to NHI it was observed that a prior inoculation of barley with Blumeria graminis f.sp. hordei (Bgh) induced susceptibility to the nonhost Magnaporthe isolate. This illustrates that the nonhost isolate, in principal, is able to infect barley. But to do this, it requires the activity of a compatible host pathogen like Bgh which can induce accessibility of plant cells to invasive pathogen growth. Thus, the nonhost isolate may only lack particular pathogenicity factors which play an essential role in the establishment of the HI. For identification of such pathogenicity factors (effector molecules) expressed by the host pathogen M. oryzae, a comparative microarray study of the Magnaporthe transcriptome during HI and NHI was performed. This led to identification of 166 effector candidate genes whose in planta expression was stronger in the host than in the nonhost isolate.In conclusion, the lists of fungal and plant candidate genes documented in the present thesis form a comprehensive basis for further dissection of nonhost resistance mechanisms. This knowledge may be exploited for breeding of new cereal cultivars with improved disease resistance in the future.