Biological Function Analysis Of MoPTEN Gene In Magnaporthe Oryzae And CgPTPM1 Gene In Colletotrichum Graminicola

Every year,pathogenic fungi pose a major threat to global food production and food security.As an important component type,the hemibiotrophic fungi,which are important constituent types,have played a huge role in the process of causing plant disease.The two stages of biotrophs and necrotrophs experienced by hemibiotrophic fungi also suggest that they have more extensive choices in nutrient utilization and host infection,and also have more regulatory pathways.Rice and Maize are the two major food crops in the world,both of which are widely cultivated worldwide.The annual yield and economic losses caused by diseases are also extremely serious.Magnaporthe oryzae and Colletotrichum graminicola are representative pathogenic fungi of rice and maize crops.Recent studies have shown that since the two fungi are both hemibiotrophic types,there are some similarities in the pathogenesis and infection mechanism,but in some biological characteristics,specific infection methods and pathogenic development processes there are also significant differences.Up to now,the research on the pathogenic mechanism of M.oryzae has made great progress,while the research on C.graminicola is still relatively lagging behind.The processes of phosphorylation and dephosphorylation of cells in organisms are closely related to various life processes such as cell development,differentiation,and signal transduction.This process is in a dynamic equilibrium state in eukaryotic cells,which is the result of the combined action of protein kinases(PKs)and protein phosphatases(PPs).At present,studies have shown that PKs play an important role in the growth and development,spore production,and pathogenicity of pathogenic fungi,but the role of PPs in the above processes is still not very clear.Based on the previous research,our laboratory focused on the function of the protein tyrosine phosphatase gene homologous to the human PTEN gene in M.oryzae and C.graminicola,in order to analyze the mechanism of this PTP in regulating the physiology and pathogenicity process of the two pathogenic fungi.In this study,we used H₂O₂ as a screening medium in a rice blast fungus insertion mutant library constructed in our laboratory to obtain a strain S28515 that is sensitive to H₂O₂ and has reduced pathogenicity.The gene of mutant was analyzed to encode a putative protein phosphatase Mo PTEN.Bioinformatics analysis showed that the phosphatase belongs to the PTPs family,has a conserved domain and catalytic active site of the family,and enzyme activity assay showed that the protein has both protein phosphatase and lipid phosphatase activities.The gene has a high level of expression during appressorium formation and pathogenic stages,and sequencing results showed that it has alternative splicing phenomenon in different stages of M.oryzae.The results of subcellular localization suggest that Mo PTEN is not significantly expressed in the vegetative growth hypha,but it is mainly distributed in appressorium and invasive hypha.In addition,the deletion of the Mo PTEN did not affect the vegetative growth and spore morphology of the mutants,but it did affect the sporulation,the early stage of germination,and the formation of appressoria.It was also found that the mutant?Mo PTEN is more sensitive to high concentrations of H₂O₂ than the wild-type strain in the stress-resistance experiment.In the process of infection,the reactive oxygen species(ROS)generated by the host cannot be eliminated in time,which indicates that Mo PTEN may be involved in the anti-host immune response process.In the pathogenicity determination of intact rice leaves,it was found that the pathogenicity of the mutant was weaker than that of the wild type.In the drop-inoculated experiment on abraded leaves,the pathogenicity of?Mo PTEN/Mo PTEN-1 was not restored,but the situation of?Mo PTEN/Mo PTEN-2 was the opposite,which indicated that the Mo PTEN-2 was related to the pathogenicity of M.oryzae.Microscopic observation showed that most of the?Mo PTEN/Mo PTEN-1 complementation strains can form appressoria on rice leaf sheath cells,but it is difficult to further form normal invasive hyphae afterwards.Although the?Mo PTEN/Mo PTEN-2 complemention strains formed fewer mature appressoria,other normally developed conidia formed more invasive hyphae than?Mo PTEN/Mo PTEN-1.It is speculated that the two forms of splicing may have played a role in the form of relay in the infection process.In addition,in the absence of Mo PTEN,the hydrophobicity of the mutant is weakened,the cell wall of the appressorium is incomplete,the ability to produce melanin is decreased,and the mutant had less sexual reproduction ability than wild-type strain.At the same time,we compared the amino acid sequence of Mo PTEN to C.granimicola and found a protein with high homology to it.The protein was also a speculated protein tyrosine phosphatase,and we named it Cg PTPM1.Bioinformatics analysis shows that,similar to Mo PTEN,Cg PTPM1 also has a conserved domain and catalytic site of the protein tyrosine phosphatase family.Cg PTPM1 also has phosphatase activity after detection.Analysis of gene expression patterns showed that Cg PTPM1 does not have alternative splicing,but similar to Mo PTEN gene,it also has a high expression level in the generation of appressorium and pathogenicity.Biological studies had shown that the absence of Cg PTPM1 did not affect the vegetative growth and spore morphology of C.graminicola,but the sporulation of the mutant decreased significantly.The conidial germination rate decreased,the germination time was delayed,and the amount of appressoria of the mutant decreased.The results of the liquid culture experiment showed that the mutant was delayed in the formation of melanin,and the shape of the hyphae formed was more fluffy.In the pathogenic ability test,the pathogenicity of the mutant decreased,and microscopic observation showed that the number of appressoria formed during the infection was reduced.The formation of primary invasive hypha was less and delayed,and the secondary invasive hypha could not be effectively formed.The deletion of Cg PTPM1 made the mutant more sensitive to H₂O₂.The hydrophobicity of mutant was weakened,and the inhibition by Congo Red and SDS was stronger,which proved that the cell wall integrity of the mutant was damaged.Subcellular localization showed that Cg PTPM1 was present in the mitochondria of C.graminicola.In summary,we compared the putative PTPs genes Mo PTEN and Cg PTPM1 in M.oryzae and C.graminicola.The results show that the phosphatase genes are related to the physiological processes of conidiation,appressorial formation and pathogenicity of the two pathogenic fungi,and participates in the hydrophobicity and the maintenance of the integrity of the cell wall.Meanwhile,it is also involved in the process of pathogenic fungi responding to the host's defense response.Our research results show the role of PTPs in hemibiotrophic ascomycetous fungus,and also provide a certain theoretical reference for drug design targeting PTP gene regulatory pathways.Therefore,it has important scientific significance.