The Synergistic Effect Of Amino Acid Permeases And Aspartate Aminotransferases In Pathogenicity Of Magnaporthe Oryzae And Colletotrichum Graminicola

The planting area of rice and corn accounts for 60% of the total area of grain planting,they are the major crops in my country.Rice blast caused by Magnaporthe oryzae is the most severe fungal disease on rice.Rice blast can lead to serious reduction of rice production and cause great economy losses.Corn anthracnose caused by Colletotrichum graminicola has become more and more serious.As semi-biotrophic fungi,M.oryzae and C.graminicola have certain similarities in infection and pathogenicity.The infection process of by semi-biotrophic fungi includes biotrophic stage and necrotrophic stage.M.oryzae and C.graminicola usually face nitrogen-deficiency during the biotrophic stage of infection.The study of amino acid absorption and metabolism of M.oryzae and C.graminicola is crucial for analyzing the pathogenic mechanism of the two pathogens.Amino acid permease and aspartate aminotransferase play important roles in amino acid metabolism in many organisms,but the cooperative between amino acid absorption pathway and amino acid synthesis pathway is still unclear.In this study,using M.oryzae and C.graminicola as experimental materials,we performed phytopathology and molecular genetics studies on Amino acid permease and aspartate aminotransferase,in order to confirm the synergism between Amino acid permease and aspartate aminotransferase.Using the known amino acid permease gene sequence of yeast as query sequences,we identified 21 candidates from M.oryzae.Using the candidates from rice blast fungus,we identified 21 candidates from corn anthracnose.The expression levels of amino acid permease genes from M.oryzae and C.graminicola at different stages were analyzed,it was found that different amino acid permease genes played roles in different stages of the pathogenic process.At the same time,based on yeast aspartate aminotransferase gene sequences,3 rice blast aspartate aminotransferases and 3 corn anthracnose aspartate aminotransferases were identified,Moast2 and Cgast2 genes may play a role in the necrotrophic stage of infection.amino acid permease and aspartate aminotransferase gene knockout mutants and complementary transformants of M.oryzae and C.graminicola were created.M.oryzae amino acid permease mutants include ?Mogap1,?Mogap2,?Mogap3,the M.oryzae aspartate aminotransferase knockout mutant was ?Moast2;the corn anthracis amino acid permease gene knockout mutant was ?Cggap3.At the same time,complementary transformants were constructed:?Mogap1/Mo GAP1,?Mogap2/Mo GAP2,?Mogap3/Mo GAP3,?Moast2/Mo AST2,?Cggap3/Cg GAP3.The biological functions of the mutants were studied.It was found that the pathogenicity of ?Mogap1 was significantly weakened,and the rice leaf sheath infection experiment showed that the conidia of ?Mogap1could infect rice,but its invasive hyphae was significantly inhibited and could not expand to adjacent cells;The pathogenicity of ?Mogap2 was also significantly weakened,and its conidia can also infect rice.The invasive hyphae of ?Mogap2 cannot obtain nitrogen sources during the necrotrophic stage,which hinders it expansion;?Mogap3 is also reduced pathogenicity,its invasive hyphae grow unaffected in the biotrophic stage and can fill the entire cell,but its extension at the necrotrophic stage was inhibited and cannot expand to adjacent cells.The pathogenicity of the?Cggap3 was also significantly reduced.Its invasive hyphae stretched slowly in the early stage of infection and were unable to infect adjacent maize cells.The pathogenicity of ?Moast2 is significantly weakened,and its infecting hyphae can expand normally in the pathogenic biotrophic stage,but in the necrotrophic stage,the invasive hyphae of ?Moast2 was inhibited.Amino acid absorption experiments show that Mogap1 could infect M.oryzae absorbing various amino acids such as Asp,Glu,Pro.Mogap3 can infect M.oryzae absorbing Asp.It indicated that Mogap1 and Mogap3 were involved in the regulation of amino acid absorption during the pathogenic process of M.oryzae.The Cggap3 is also involved in the Asp uptake of C.graminicola.It indicates that the amino acid permease of M.oryzae and C.graminicola have similar function.The amino acid metabolism experiment showed that Moast2 can catalyze the production of Glu from Asp in the rice blast fungus,then synthesizing the important amino acids such as arginine and proline for the rice blast fungus.The above results proved that the cooperation of amino acid permease and aspartate aminotransferase plays a very important role in the pathogenesis of M.oryzae and C.graminicola.