Analyse von Effektorproteinen in Interaktionen von Pilzen der Gattung Pyricularia (Magnaporthe) mit Gerste

  • Analysis of effector proteins in interactions of fungi of the genus Pyricularia (Magnaporthe) with barley

Mogga, Valerie; Schaffrath, Ulrich (Thesis advisor); Deising, Holger (Thesis advisor); Panstruga, Ralph (Thesis advisor)

Aachen (2016, 2017)
Dissertation / PhD Thesis

Dissertation, RWTH Aachen University, 2016

Abstract

Phytopathogenic fungi of the genus Pyricularia are associated with the plant family Poaceae. While major crop plants like rice, barley or wheat can be infected by P. oryzae (morph Magnaporthe oryzae), these plants are protected against isolates of the species P. grisea (morph Magnaporthe grisea) or P. penniseticola by nonhost resistance. Nonhost resistance is defined as the capacity of an entire plant species to resist infection by all isolates of a given microbe species. To successfully infect host plants, phytopathogens secrete effector molecules to circumvent or manipulate the plant immune system. The repertoire of effectors therefore contributes to host range of a pathogen. To analyze differences in effector repertoires of host and nonhost isolates from barley, transcriptomes of the M. oryzae isolate TH6772 and of the P. penniseticola isolate CD180 were compared during their interaction with barley by microarray analysis. Based on this approach, transcripts encoding small secreted effector proteins were identified that accumulate to a significantly higher extent during host interaction of barley with M. oryzae [designated M. oryzae Hypothetical Effector Genes (MoHEGs)] than during nonhost interaction with P. penniseticola. Analyses of transcript abundances during host interaction between M. oryzae and barley (0-96 h p.i.) showed that MoHEGs are differentially expressed during biotrophic interaction. Furthermore they can be differentiated into two groups according to their maximum transcript abundances (prior to or during penetration: EarlyMoHEGs or during colonization: LateMoHEGs). In contrast to LateMoHEGs, early-induced MoHEGs with expression maximum at 0 and 6 h p.i. were also transcribed during plant-independent in vitro germination of conidia. Functional analysis disclosed a crucial function for EarlyMoHEG16 during barley infection as mesophyll cell collapse was significantly reduced in barley host plants infected with three independent Δmoheg16 mutants compared to barley inoculated with the corresponding M. oryzae wild type isolate. Hence, although MoHEG16 mutation does not cause complete loss of M. oryzae virulence on barley, MoHEG16 constitutes a M. oryzae virulence factor. Additionally, LateMoHEG13 was found to suppress M. oryzae-Nep1 (Necrosis- and Ethylene-inducing Protein1)-Like Protein (MoNLP)-derived cell death in N. benthamiana. For that reason MoHEG13 may play a role in coordination of cell death induction by the fungus in general or possibly regulate other NLP-mediated responses during M. oryzae infection processes. Furthermore, putative orthologs to MoHEGs were identified in M. grisea isolate BR29 (MgHEGs) that showed a similar, MoHEG-like expression pattern during a compatible interaction. Although direct evidence has yet to be provided, these data indicate that HEG effectors might be similarly deployed by both species for successful host infection, thereby being part of an evolutionary conserved fungal infection strategy. Apart from MoHEGs another putative pathogenicity factor of M. oryzae was analyzed for its function in the M. oryzae - barley interaction. The NUDIX hydrolase MoNUDIX was functionally characterized by over-expression and silencing of the corresponding gene(s). Quantitative microscopic analysis of the mutants revealed that over-expression accelerates infection process whereas down-regulation of the gene seems to restrict pathogenic growth. MoNUDIX therefore represents a so far unknown pathogenicity factor in M. oryzae.