Biology And Pathogenic Function Of Ubiquitination-associated Factor BcUBI4 In Botrytis Cinerea

Botrytis cinerea,the causative agent of plant gray mold,is a typical necrotrophic phytopathogenic fungal pathogen that infect more than 1000 different plant species and causes huge economic losses in agricultural production.B.cinerea can infect different organs of plants,causing damage at all stages of growth,Botrytis cinerea mainly infect the host via its conidia.conidia contact with the host,will be regulated by a series of signals to induce conidia germination to generate budding tubes,attached spores,infestation mats to invade the host cells,Botrytis cinerea also degrade the host cell wall by secreting various types of cell wall degrading enzymes,small molecule toxins to kill the host and obtain nutrients to to meet their own growth and development.Nevertheless,our understanding of the pathogenicity-related factors of Botrytis cinerea and how they regulate the pathogenicity of the bacterium remains to be improved.Ubiquitin is a highly conserved protein consisting of 76 amino acids,and the process of ubiquitination refers to the specific modification of a class of proteins with small molecular mass by the action of specific enzymes such as activating,binding,ligating and degrading enzymes.At the same time,it is involved in the regulation of nearly all life activities such as cell differentiation,gene expression,transcriptional regulation,signaling and pathogenesis.There are many pathogenicity-related genes involved in the pathogenesis of Botrytis cinerea,but there are still many genes whose functions are unknown,and there is a lack of studies about the ubiquitination protein UBI4 in regulating pathogenic development,pathogenicity and pathogenesis of plant pathogenic fungi,especially Botrytis cinerea.This study focuses on the role of the BcUBI4 gene,which encodes a ubiquitination protein,in the pathogenesis of Botrytis cinerea.We obtained a mutant strain M22017 with weak pathogenicity by screening the transformant population containing about50,000 T-DNA inserts constructed in our laboratory,and found that the T-DNA was inserted into the genome of Botrytis cinerea encoding ubiquitin protein gene by TAILPCR(thermal asymmetric interlaced PCR).BcUBI4 upstream.To investigate the function of BcUBI4,the deletion mutants ΔBcubi4-1 and ΔBcubi4-2 of the BcUBI4 gene of Botrytis cinerea and the back-complementary transformants ΔBcubi4-C were obtained using homologous recombination and Agrobacterium-mediated genetic transformation methods in this study,and pathogenicity assays revealed that the BcUBI4 gene affects the pathogenic process of Botrytis cinerea.By comparing the growth,ability to infest hosts and stress response to adversity between wild-type B05.10 and BcUBI4 gene deletion mutant strains,we found that the BcUBI4 gene deletion mutant showed significant differences during growth and at the beginning of infestation,as follows.1.In this study,we phenotypically analyzed the wild-type,BcUBI4 deletion mutant and back-supplemented strains of Botrytis cinerea,and firstly,we found that the mutant strains were significantly stunted in growth compared to the wild-type and backsupplemented strains of Botrytis cinerea.The deletion of BcUBI4 severely affected the normal growth process of the mutant in mycelial elongation,infestation mat formation and nucleus germination.The results of pathogenicity-related experiments showed that the UBI4 deletion mutant did not produce conidia and could not form complete infestation structures to invade the host plant cells,and almost completely lost pathogenicity.This suggests that BcUBI4 may regulate the pathogenicity of Botrytis cinerea by regulating the pathogenesis-related developmental processes of the pathogenic bacteria.2.In an investigation of the possible involvement of BcUBI4 in regulating the response of Botrytis cinerea to adversity stress,several different exogenous substances were added to CM medium,including B05.10,ΔBcubi4-1,ΔBcubi4-2 and ΔBcubi4-C by adding 0.005% SDS,0.01% SDS and 600 μg/m L of Congo red to the culture The results showed that BcUBI4 is likely to be involved in the cell wall synthesis process of Botrytis cinerea.Inhibition of ubiquitin-mediated protein hydrolysis using the exogenous 26 S proteasome inhibitor bortezomib significantly inhibited conidial germination,adherent cell formation and pathogenicity in Botrytis cinerea,and the results were more consistent with the experimental phenotype of the UBI4 deletion mutant.In addition,the expression of UBI4 gene was significantly inhibited when wildtype strain B05.10 was cultured on plate medium containing bortezomib,further corroborating that UBI4 regulates the synthesis of the cell wall of Botrytis cinerea by participating in the ubiquitination process,which in turn affects the subsequent pathogenicity-related development and pathogenicity of Botrytis cinerea.Therefore,we conclude that the ubiquitination-related regulation of cellulose synthesis and degradation by BcUBI4 may be an important way for Botrytis cinerea to participate in the invasion process of pathogenic bacteria.The present study systematically resolved the biological and pathogenic functions of the BcUBI4 gene of Botrytis cinerea,and the results of the study contributed to our understanding of the role played by BcUBI4 pathogenicity-related factors in plant pathogenic fungi,and also enabled the targeting of the possible role of BcUBI4 in the ubiquitin pathway,which provides potential new molecular targets for the development of new drugs against important plant pathogenic fungi It also provides a scientific basis for the development of new strategies for the prevention and control of gray mold.