| Cassava(Manihot esculenta Crantz)is a kind of food crop and energy plant widely planted in the tropics and subtropics.It is drought tolerant and can grow in low-fertility soils.However,its yield is seriously constrainted by pathogen infection.Therefore,elucidating the molecular mechanism of defense response involved in cassava-pathogen interaction become an important issue.Reactive oxygen species(ROS)generating systme play an important role in plant disease resistance.The NADPH oxidase encoded by Rboh(Respiratory burst oxidase homologue)is one of the main sources of ROS.In this study,the role of Rboh gene family in cassava disease resistance was investigated for the first time through genome-wide identification,bioinformatics analysis,subcellular localization,expression pattern analysis and gene function verification.The main results are as follows:(1)Eight Rboh genes were identified from cassava genome,named MeRbohA-H.The amino acids encoded by MeRbohs ranged from 845-954,the relative molecular weight ranged from 96.0-108.7 KDa,and the isoelectric point range were 9.09-9.41.The conserved domain analysis showed that all the 8 MeRBOHs had the characteristic domains of RBOH which were two Ca2+ binding EF-chiral structures at N-terminal,six transmembrane domains(TMD I-TMD VI)and FAD and NADPH hydrophilic domains at C-terminal.Phylogenetic analysis showed that RBOHs from cassava,Arabidopsis thaliana and rice could be classified into three subfamilies.Gene structure analysis showed that the numbers of introns of MeRbohs ranged from 12-14,and the chromosome localization analysis indicated that,except for MeRbohG,the other seven genes were distributed on 7 different chromosomes.The results of promoter analysis showed that the promoter region of MeRbohs contains many regulatory elements involved in growth and stress signal pathway.(2)The results of subcellular localization in Arabidopsis protoplasts showed that 8 MeRBOH proteins were plasma membrane localized,which were consistent with the localization of NADPH.(3)The results of qRT-PCR showed that the expression patterns of MeRbohs in cassava roots,stems and leaves were different.MeRbohA,MeRbohD,MeRbohG and MeRbohH were highly expressed in roots,stems and leaves,MeRbohB,MeRbohE and MeRbohF in roots,while MeRbohC mainly expressed in roots and leaves.The expression level of MeRbohs were induced by H2O2,cassava pathogen Xam,salicylic acid and jasmonic acid treatments,except the MeRbohC expression was inhibited by salicylic acid.(4)The weakened PTI pathway in Arabidopsis mutants rbohB,rbohD and rbohF could be restored by complementation of MeRbohB,MeRbohD and MeRbohF respectively.(5)The results of Pst DC3000 infection experiments showed that the Arabidopsis rbohD mutants were hypersensitive to pathogens infection.The accumulation of peroxide and callosum in infected leaves of mutant was significantly lower than that in wild type,while in complementaion and overexpression plants,these phenotypes were partially restored.rbohF mutant was more sensitive to pathogen infection,but the accumulation of peroxide and corpus callosum was not changed compared with wild type.However,these phenotype was restored in transgenic plant by overexpressing MeRbohD;although the susceptibility of Arabidopsis rbohD mutants to pathogen Pst DC3000 was also boosted,the accumulation of peroxide and corpus callosum was not changed in comparation with wild type.At the same time,the accumution were increased in the complementation and overexpressed transgenic Arabidopsis.The phenotypes of rbohB mutants,complementary and overexpressed plants were in consistent with wild type. |
