Molecular Mechanism Involved In Oxidative Stress Tolerance And Virulence To Postharvest Fruit Mediated By The Peroxiredoxin PePrxV1 Of Penicillium Expansum

Penicillium expansum is a plant fungi pathogen widely distributed in nature worldwide,which could cause severe decay and mycotoxin contamination in many postharvest cereal or fruit crops.Apple fruits,as popular fruits in world market,have multiple valuable nutrients and sweet taste.However,postharvest apple fruits are easy to be infected by pathogens,resulting in huge economic losses.P.expansum is one of the main fungi pathogens causing apple fruits’decay.The current physical control methods could delay but not completely inhibit the growth of P.expansum.Biological control methods provide promising alternatives,but their cost is high and their application is limited.Therefore,it is necessary to further reveal the molecular mechanism of infecting hosts for P.expansum.For successful infection,P.expansum not only enhances its own pathogenic ability,but also inhibits the expression levels of defense related genes in plant hosts,during the whole infection process.With the emergence of omics and molecular biology technologies and methods,such as whole genome sequencing and targeted gene deletion,it would help us to better understand the molecular mechanism of infecting plant hosts for P.expansum.H₂O₂ stress tolerance is an important factor for the survival and success of P.expansum during infecting plant hosts.Whereas,little is known about Peroxiredoxin（Prx）in P.expansum.In order to explore the roles of Prx in P.expansum in H₂O₂ tolerance and virulence,Prx family genes were identified from genome sequences of P.expansum.Based on phylogenetic analyses and H₂O₂ induced expression levels,one Prx V subfamily gene,Pe Prx V1（Gen Bank accession number:PEX2＿104300）,in P.expansum was selected to construct knockout mutants for further gene function illustration.The specific results of the study are as follows:（1）A total of six Prx genes were identified in P.expansum genome,distributed in three subfamilies,BCP-Prx Q,PrxⅤand PrxⅥ.Three Prx genes belong to PrxⅤsubgroup,including PEX2＿104300（PePrxⅤ1）,PEX2＿063590（Pe PrxⅤ2）and PEX2＿091510（Pe PrxⅤ3）.Bioinformatics analyses of the protein sequences of PePrxⅤ1,Pe PrxⅤ2 and Pe PrxⅤ3 showed that PePrxⅤ1 was significantly different from Pe PrxⅤ2 and Pe PrxⅤ3in protein sequence composition,physical and chemical properties and three-dimensional structure,respectively.（2）The knockout mutants of PePrxⅤ1（ΔPe Prx V1）in P.expansum were constructed,and phenotypically analyzed.Under H₂O₂ stress,spore germination ofΔPe Prx V1 on potato dextrose broth（PDB）medium was delayed than that of wild type.On potato glucose agar（PDA）medium plate containing H₂O₂,the growth rate was also slower than that of wild type.In addition,scanning electron microscopy assay showed that H₂O₂stress caused more serious damage inΔPe Prx V1 mycelium than that in wild type.Fluorescent images of Rhodamine 123 staining showed lower fluorescence intensity inΔPe Prx V1 mycelium under H₂O₂ stress,indicated more decrease of mitochondrial membrane potential,and more apoptosis in response to H₂O₂ stress.Furthermore,under H₂O₂ stress,the content of reactive oxygen species（ROS）and malondialdehyde（MDA）inΔPe Prx V1 cells increased,and the content of adenosine 5’-triphosphate（ATP）as well as the activities of peroxidase（POD）and catalase（CAT）decreased,indicating that Pe Prx V1 gene’s knockout weakened the ability of scavenging ROS,and resulted in more severe mycelial damage and mitochondrial apoptosis.（3）After treatment with 25 mmol L^-1H₂O₂ for 5h,the expression profiles of Prx,CAT,Kat,GPx,APX and Dy Prx＿D,mainly responsible for ROS scavenging system,were determined by quantitative real-time PCR（q RT-PCR）in wild type andΔPe Prx V1.The results demonstrated that both up and down expression tendencies existed in the same family members in comparison with wild type.The increased expression levels of some genes may be a supplement to the knockout of Pe Prx V1 and the decreased expressions of other peroxidase-encoding genes.However,under H₂O₂ stress,this supplement mechanism is not enough to restore POD activity to a normal level.（4）The spores of wild type andΔPe Prx V1 were inoculated in apple fruits to evaluate their pathogenicity.The results showed thatΔPe Prx V1 reduced the diameter of decay on apple fruits by about 20%in comparison with wild type after three days’infection.Furthermore,MDA content in apple fruits inoculated withΔPe Prx V1 was lower than that of apple fruit infected with wild type.Meanwhile,the activities of POD and CAT in apple fruits infected withΔPe Prx V1 were higher than those in apple fruits infected with wild type.（5）The P.expansum-apple interaction RNA-seq was performed in apple fruits inoculated with wild type andΔPe Prx V1.The expression profiles of pathogenic effector genes in the transcriptome revealed the decreased or increased levels of 31 effector genes.The transcriptome data of nine selected effector genes were verified by q RT-PCR.Our data suggested that Pe Prx V1 may affect the virulence of P.expansum through up/down-regulate the effector genes.Additionally,the expression profiles of JA biosynthesis,signal transduction genes and PR genes in apple fruits infected with wild type andΔPe Prx V1 were compared.Most genes involved in JA biosynthesis,signal transduction and PR genes showed an upward trend in apple fruits infected byΔPe Prx V1.Whereas,PAL（MD12g1116700）,a key gene in upstream of SA synthesis,showed decreased expression levels in apple fruits inoculated withΔPe Prx V1 in compared with those inoculated with wild type.The results of q RT-PCR further showed the consistent trends with the transcriptome data,indicating a stronger JA-regulated defense response in apple fruits inoculated withΔPe Prx V1.In summary,Pe Prx V1 plays a key role in spore germination,mycelial growth,H₂O₂ stress tolerance and virulence to postharvest fruits in P.expansum.