The Role Of GmNIG1 In Soybean Resistance To Soybean Cyst Nematode

Soybean cyst nematode(Heterodera glycines Ichinohe,SCN)disease is a worldwide problem of soybean.Nowadays,traditional chemical prevention and treatment no longer meets the requirements of sustainable development of agriculture,and it is necessary to explore effective and environmentally friendly strategies.Planting disease-resistant varieties is one of the most cost-effective measures.However,due to the adaptaion of SCN to the resistant cultivars and the increase of virulent nematode populations,the development of resistance resources against multiple races is particularly important.In previous study,a nematode inducible gene Gm NIG1,which belongs to the pathogenesis-relataed 10 gene family,was obtained through transcriptome data analysis of the susceptible variety Williams 82(W82)and the resistant variety PI88788 infected with SCN combined with promoter-GUS verification.The soybean hairy root transient transformation system was used to analyze the function of Gm NIG1.Overexpression of Gm NIG1 revealed that Gm NIG1 positively regulates soybean resistance to soybean cyst nematode.Yeast two-hybrid(Y2H)screening obtained ethylene response factors group VII proteins(ERFVIIs)as interacting candidates.The interaction between Gm NIG1 and three members of Gm ERFVIIs was further confirmed using Y2 H,bimolecular fluorescence complementary(Bi FC)experiment,luciferase complementary imaging(LCI)experiment,and co-immunoprecipitation(Co-IP)assay,and the interaction occurs in the nucleus.To understand the interaction mechanism betweenthe two proteins,we investigated the biochemical mechanism of Gm NIG1 and three members of Gm ERFVIIs.Gm ERFVIIs were truncated to different lengths and the transactivation domain and Gm NIG1-interacting domain were analyzed.We found that Gm ERFVIIs has transactivation activities with two transactivation domains confirmed and AP2 domain is required for the interaction with Gm NIG1.Subcellular localization shows that Gm NIG1 and Gm ERFVIIs are localized in the cytoplasm,cell membrane,and nucleus.Substitution of N-terminal residues which were proved to be critical for the N-degron pathway can stabilize Gm ERFVII proteins and regulate the nucleus localization.Western blotting results with the proteasome inhibitor MG132 confirmed that 26 S proteasome pathway is involved in the regulation of destabilization of Gm ERFVIIs.Recombinant Gm NIG1 protein shows RNase activity but not DNase activity.q RT-PCR results showed that the expression of Gm NIG1 and Gm ERFVIIs was induced after SA and JA treatment.Since Gm ERFVIIs function in oxygen sensing pathway,we tested the expression levels of disease-related genes(such as PR1,PR4,etc.)and hypoxic-responsive genes(such as ADH,PDC,etc.)in Gm NIG1 overexpression transgenic hair roots.We found that Gm NIG1 can positively regulate the expression of genes related to disease resistance and hypoxic response.In summary,these results suggest that Gm NIG1 plays an important role in contributing to soybean resistance to soybean cyst nematode by regulating Gm ERFVIIs-mediated oxygen sensing pathway,but the underlying mechanism needs to be further elucidated.These results will provide us theoretical support for studying the mechanism of soybean defense responses to nematode.