The Role Of Polygalacturonase And Pectate Lyase In Pathogenicity Of Colletotrichum Gloeosporioides And The Regulation Mechanism Of DNA Methylation

Mango(Mangifera indica L.)is an important economic crop in China.Anthracnose caused by Colletotrichum gloeosporioides(Penz.)is one of the main diseases,which leads to serious economic losses of mango during storage and transportation.DNA methylation is an important epigenetic mechanism of eukaryotes,which involved in the regulation of gene expression in animals and plants under the catalysis of methyltransferase(DNMT).However,the relationship between the process of fungal infection and DNA methylation has not been studied.In order to investigate the changes of pectinolytic enzymes activity level of C.gloeosporioides and the pathogenic mechanism of mango infection,the activity levels of polygalacturonase(PG),pectate lyase(PL)and DNA methyltransferase of C.gloeosporioide were measured in vivo mango and inoculated on different culture media,The colony morphology(disease symptoms)and its rate of expansion were observed and determined.Furthermore,the methylation sites of Cp G island in above pectinolytic enzymes gene were screened,the changes of methylation sites and DNA methylation levels of Cp G island were detected by bisulfite sequencing PCR,and the changes of m RNA transcription level of target gene were detected by quantitative PCR technology.So as to further analyze the regulation of DNA methylation on pectinolytic enzymes gene in the process of C.gloeosporioides infection.The results are as follows:(1)During the inoculation period,the polygalacturonase and pectate lyase activity level of C.gloeosporioides increased first and then decreased.The above pectinolytic enzymes activity levels of C.gloeosporioides increased significantly after inoculating on pectin agar medium and mango fruit tissue agar medium.These enzymes activity levels of the lesion part reached a high level on 3 d and 5 d after inoculation in vivo mango with C.gloeosporioides.(2)The growth rate of C.gloeosporioides colony in different medium increased first and then decreased.The expansion rate of lesion area increased after inoculation in vivo mango.The growth rate was higher at 3-5 d after inoculation.(3)The gene expression levels of PG₁,PG₂and PL in C.gloeosporioides after inoculating on different media and in vivo mango were as follows:compared with the initial level of gene expression,PG₁,PG₂,and PL gene expression level of C.gloeosporioides was significantly up-regulated on 7 d inoculated on different medium.In addition,PG₁,PG₂and PL gene expression levels of C.gloeosporioides on 3 d up-regulated significantly inoculated on mango fruit tissue agar medium.However,after inoculating in vivo,PG₁,PG₂and PL gene expression levels of C.gloeosporioides significantly up-regulated on 3-5 d.(4)The DNMT enzyme activity and DNMT gene expression level of C.gloeosporioides inoculated on different media and in vivo showed the same trend,which decreased at first and then increased during inoculation.And these maintained a low level on 3-5 d.(5)The DNA methylation level of Cp G islands screened by PG₁,PG₂and PL genes of C.gloeosporioides was generally lower on 3 d,and higher on 0 d and 7 d.The DNA methylation level was positively correlated with the changes of DNMT expression level,and was negatively correlated with the m RNA regulation level of PG₁,PG₂,and PL genes of C.gloeosporioides during infection.According to the above results,polygalacturonase and pectate lyase may be involved in the infection process of C.gloeosporioides,which provides a theoretical basis for the mechanism of pectinolytic enzymes in the pathogenic process of fungi.In addition,DNA methylation of C.gloeosporioides negatively regulated on the expression of pectinolytic enzymes gene under the catalysis of DNMT,which suggests that DNA methylation may be involved in the regulation of fungal infection.These provides new evidence for the epigenetic mechanism in the infection procedure of C.gloeosporioides.