Functonal Characterization Of GmbZIP44 In Soybean And Soybean Cyst Nematode Interaction

Soybean（Glycine max）is an important grain and oil crop.It is also the largest source of feed in the word.The soybean cyst nematode（Soybean Cyst Nematode,SCN）is widely distributed in the main soybean producing areas of our country,which poses a great threat to soybean production.At present,the most effective control method is to select and plant resistant varieties.However,the successive planting of resistant varieties will lead to the emergence of new and highly virulent physiological varieties.Therefore,it is particularly important to find new resistance genes and it is also very important to further analyze the molecular mechanism of soybean resistance to SCN.The SNF1-related protein kinase SnRK1 and the C/S1-bZIP transcription factor are key signaling regulatory components in plant carbon and nitrogen metabolism and energy balance.The previous phosphoromic data in our laboratory indicated that the phosphorylation level of a GmbZIP44 transcription factor in soybean is regulated by soybean cyst nematodes.This study mainly focused on the function of GmbZIP44 and the regulatory mechanism of its upstream and downstream signaling pathways in soybean cyst nematode resistance.In the experiment,the function of its upstream kinase GmSnRK1 and the regulation signaling pathway of downstream target gene in resistance studies was explored.According to the previous phosphorylation group data,we found when R3 infected the root of soybean W82,the phosphorylation level of GmbZIP44-04g was significant up-regulated.The phosphorylation level of GmbZIP44-04g was up-regulated 7.06times in susceptible variety W82.Subcellular localization results show that GmbZIP44is mainly located in the nucleus.GmbZIP44 can form homodimers and form heterodimers with GmbZIP63 in yeast.GmbZIP44-04g and its phosphorylation site to form active and inactive mutants(GmbZIP44-04g^S66D and GmbZIP44-04g^S66A)were overexpressed in soybean hairy roots during the experiment.The results of disease resistance phenotype experiments showed that the overexpression of GmbZIP44 could enhance soybean resistance to soybean cyst nematodes.The overexpression of GmbZIP44-04g ^S66D was more significant in disease resistance.The overexpression of GmbZIP44-04g ^S66A showed obvious susceptibility phenomenon.These results indicated that GmbZIP44 and its serine site were involved in the regulation of soybean cyst nematode resistance.GmSnRK1 can directly interact with GmbZIP44 by yeast two-hybrid,luciferase complementary imaging（LCI）and GST co-immunoprecipitation assay.Subcellular localization showd that GmSnRK1 localizes to the cytoplasm and nucleus and it colocalizes with GmbZIP44 in the nucleus.Overexpression of GmSnRK1 showed disease resistance.Consistently,the gene silencing of GmSnRK1 resulted in significantly shorter soybean roots and increased susceptibility to cyst nematodes.The analysis of qRT-PCR revealed that the key metabolic genes GmDIN1-01g,GmASN1-02g,GmDIN10-14g and GmProDH1-18g related to low energy response were regulated by GmSnRK1 and GmbZIP44.It was found that the developmental progress of soybean cyst nematode was blocked when DCMU and dark treatment were used to simulate the starvation state of plants.These results suggested that low-energy stress response signaling pathways may be involved in soybean cyst nematode resistance.