Study On The Relationship Between Mic60 Gene And JA/ET Signaling Pathway In Resistance Of Arabidopsis Thaliana To Botrytis Cinerea

Botrytis cinerea can infect more than 200 crop species.And it is considered as one of the most important fungal plant pathogen in the world.The infection of B.cinerea causes crop outbreak of mould gray disease by infecting crops and causes serious economic losses.In the previous research in our lab,a T-DNA inserted mutant that displayed enhanced sensitivity to Botrytis cinerea was obtained in Arabidopsis thaliana by genetic screening.The mutant was named as esb1(enhanced susceptibility to Botrytis1)and the corresponding gene was designated as ESB1.The mutant gene was localized on AT4G39690.The previous research results showed that knockout line for this gene,esb1-1 was susceptible not only to infection of Botrytis,but also to stresses of salt,drought and oxidation after ESB1 mutation.The patterns of gene expression studies indicated that ESB1 was constitutively expressed in Arabidopsis plants.However,its expression could be induced by Botrytis and salt stress also.Our previous research also suggested that resistant function of ESB1 may be related with hormone signaling pathways.Michaud et al.found that AT4G39690 encodes a component(named as AtMic60)of mitochondrial transmembrane lipoprotein complex(MTL)in Arabidopsis plants in 2016.The MTL complex contains two proteins,such as AtMic60 and TOM.AtMic60 is located in the inner mitochondrial membrane and conserved in all eukaryotic cells.They showed that Mic60 played a key role in mitochondria membrane biogenesis probably by lipid trafficking,thus we changed the name of ESB1 to Mic60,and esb1-1 to mic60.2 according to the principle of first publication of gene's name.Based on these data,the mitochondria morphology of atmic60.2 and mutant complementation line of atmic60.2 HB-10 were observed by electron microscopy in this study.The alterations of mitochondria morphology were observed.The sizes of mitochondria in atmic60.2 were bigger than that in wild type.The shape was changed to more round.However,it was not observed that the typical “onion like structure” of mitochondria inner membrane caused by the mutation of the humongous gene Mic10 in yeast.The shapes and the sizes of mitochondria in the genetic complementary plant HB-10 are similar with that in the wild-type plants.In this experiment,the responses of mic60.2 complementation line HB-10 and Mic60 gene overexpression line OE-17 to Botrytis were determined.The results showed that compared with the wild-type plant,the resistant level of HB-10 line to the fungal infection was restored to that of the wild-type plant.And the resistance to the pathogen of OE-17 line was obviously increased comparing with wild-type plants.These experiments further verify that Mic60 gene is played a key role in the resistance to B.cinerea in Arabidopsis.To understand the relationship between the Mic60 and the hormone signaling which associating with disease resistance,the gene expressional level was examined by real-time quantitative RT-PCR from following three respects.First,expressional levels of the important hormone signaling node genes,such as JAR1,COI1 for JA pathway,EIN2 for ET pathway,and NPR1 for SA signaling were checked in mic60.2.The results showed that the expressional levels of four genes in the mic60.2 were obviously reduced.It may imply that the Mic60 gene was associated with the JA,ET and SA signaling pathways.Secondly the expression of Mic60 in the mutants of jar1?coi1?npr1?noa1 and abi4 were quantitatively analyzed.The result showed that the expression of Mic60 in the mutants of jar1 and npr1 were reduced apparently.The third,the expressional quantity of JAR1,COI1?EIN2?NPR1 were measured after inoculation with Botrytis for 24 h in mic60.2 plant.The data showed that the expressional level of JAR1,COI1?EIN2?NPR1 were decreased remarkably.But the expressional level of NPR1 which related to SA pathway had no significant difference after inoculation with pathogen.Those results may imply that the Mic60 mediate the resistance of Arabidopsis to B.cinerea though the JA/ET hormone signaling.In this study,a double mutant of atmic60.2 jar1 was constructed by hybridization.And the homozygositic double mutant mic60.2 jar1 was obtained.It will provide the materials for further study.