The Novel Gr29D09 Effector Family From Potato Cyst Nematode Globodera Rostochiensis Suppresses Plant Immunity To Promote Nematode Parasitism
Potato cyst nematodes - Globodera rostochiensis and G. pallida are considered to be the most devastating plant pathogens that threaten the potato production in the United States and worldwide. Understanding the functions of nematode-secreted effectors is necessary to uncover the molecular mechanisms of nematode parasitism which could be translated into novel nematode control strategies. The novel 29D09 gene was cloned as a candidate effector gene in the soybean cyst nematode Heterodera glycines and in G. pallida. An initial study confirmed the expression of 29D09 orthologues (Gr29D09) in G. rostochiensis. The overall objective of this study was to clone and characterize the function of Gr29D09 genes in G. rostochiensis parasitism. Specifically, this thesis focuses on four research objectives: i) To clone and analyze Gr29D09 sequences from G. rostochiensis populations, and to investigate their roles in nematode parasitism; ii) To determine a role of Gr29D09 effectors in host innate immunity suppression; iii) To identify Gr29D09-targeted proteins in potato; and iv) To develop PTI assays that can be used in plant-nematode pathosystems. Sequence analysis revealed that Gr29D09 belongs to a gene family because multiple gene sequences were identified in individual nematodes. Gr29D09 genes were found to be exclusively expressed within the dorsal gland cell and significantly upregulated in nematode parasitic stages, suggesting an involvement of this effector family in nematode parasitism. Polymorphic variants of Gr29D09 family were indicated to have undergone diversifying selection, suggesting that this effector family has evolved to avoid host immune system. Multiple approaches were utilized to investigate the function of four major Gr29D09 variants. Gr29D09 overexpression in potato resulted in increased susceptibility to G. rostochiensis and an unrelated potato pathogen Streptomyces scabies, as well as the suppression of callose deposition after treatment with PTI inducer Pseudomonas fluorescens. Using Agrobacterium-mediated transient expression assays in Nicotiana benthamiana, we found that all four Gr29D09 variants were able to suppress PTI and ETI responses to variable degrees. These results clearly demonstrated a critical role of Gr29D09 effectors in suppression of plant immunity, possibly by involving different defense signaling pathways. Interestingly, transient expression of two Gr29D09 variants (Gr29D09-V3 and Gr29D09-V4) were found to induce hypersensitive response in leaves of two wild potato species and the potato cultivar 'Kennebec', indicating that the two variants may function as avirulence proteins for yet-to-identified R proteins in these potato species. Co-immunoprecipitation coupled with nano LC-MS/MS analysis identified three defense-related proteins Hexokinase 1, heat shock protein 90 and a homologue of the late blight resistance protein R1A as strong candidate host targets of Gr29D09-V1 and Gr29D09-V3. Altogether, this study demonstrated that G. rostochiensis uses Gr29D09 effectors to suppress plant innate immunity to promote successful nematode parasitism.
Gr29D09, potato cyst nematode; Globodera rostochiensis; suppresses plant immunity
Martin,Gregory B; De Jong,Walter S.
Ph.D. of Plant Pathology
Doctor of Philosophy
dissertation or thesis