The Biological Roles Of Both General Amino Acid Permeases MoGAP2 And MoGAP3 In Magnaporthe Oryzae

The rice blast caused by Magnaporthe oryzae is an important disease on rice,causing serious economic losses worldwide every year.The study of its pathogenic mechanism,as a model,also has important reference significance for the prevention and control of fungal diseases on other crops.The utilization of nitrogen,synthesis and transportation is related to the normal operation of biological functions of lives and stability of the internal environment.As a component of protein,amino acids directly participate in a variety of physiological and biochemical processes in the fungus cells,and some amino acids are also used as functional sites for binding or modification between proteins,which are essential to the various life activities of M.oryzae.There have been mang studies on the key enzyme genes in the amino acid synthesis pathways such as methionine,isoleucine,lysine and aspartic acid,but there are few research on the amino acid transport of rice blast fungus.Our subjects mainly explore the biological functions of three general amino acid permeases and their possible relationships,and identify the roles of three permeases in the growth,development and pathogenesis of M.oryzae.In the previous experiments we have obtained mutant strains△MoGAP1 and△MoGAP3 that lack amino acid permease 1 and 3 genes（MoGAP1,MoGAP3）through gene knockout experiment,and analyze the biological function of MoGAP1in M.oryzae.I obtained the deletion mutant△MoGAP2 of general amino acid permease 2 gene（MoGAP2）through ATMT method,and verified the functions of MoGAP2 and MoGAP3 at the same time,in order to figure out the impact of deletion of permease on the biological characteristics of M.oryzae and the association among the three permeases,the following results are obtained:1.Through bioinformatics analysis,the full length of the MoGAP2 is 2626bp,without intron sequence,and the open reading frame encodes 576 amino acids.MoGAP3 has a total length of 2205 bp and contains a 75 bp intron,which encodes554 amino acids.Predicting the protein structure of MoGAP2 and MoGAP3,it is found that both of them belong to the Lys P protein family,and contain an amino acid permease domain（AA permease）.Unlike MoGAP1,MoGAP2 and MoGAP3 both contain 12 transmembrane domains.There are 13 in MoGAP1.2.Compared with wild-type Y34,the vegetative growth and germination of△MoGAP1 are reduced,and the number of conidia is also reduced,which is consistent with Song Yanyue’s experimental results.But the differences among△MoGAP2,△MoGAP3 and Y34 are not significantly.The appressorium morphogenesis of three mutants is not different from that of the wild type.3.The pathogenicity of△MoGAPs in rice leaves and leaf sheaths was weakened.The gene expression pattern during the infect process show that when the fungi infects rice leaves,the expression of MoGAP1 and MoGAP2 upregulate over time,while the expression of MoGAP3 is up-regulated within 3 days and then gradually decreased.It shows that the general amino acid permease genes are related to the pathogenicity of M.oryzae.4.Using 3,3-diaminobenzidine（DAB）staining solution to label the hydrogen peroxide produced by rice leaf sheath cells,we found that the mutant was subjected to stronger oxidative stress from the host cell during the infection process,making its ability to infect neighboring cells is inhibited.In the H₂O₂ stress experiment,the expression of permease gene indicating that the three general amino acid permease genes were involved in the pathogenic process of M.oryzae,especially in the response to oxidative stress at the initial stage of the infection event,and these three permeases shows partly synergy during these processes.5.The growth rate and trend of the three mutants in the media supplemented with H₂O₂ are similar to those of the wild type,and have not been significantly affected.Adversity stress experiments show that MoGAP2 and MoGAP3 are sensitive to osmotic stress from Na Cl.Congo red and SDS experiments show that MoGAPs also have a positive effect on the integrity of cell walls.6.In the minimal media lacking any nitrogen source,the growth rate and biomass of△MoGAP1 were significantly reduced.△MoGAP3 was reduced,but not significant enough.q PCR analysis found that the expression of MoGAP1 and MoGAP3 was significantly up-regulated,indicating that the two permease genes may play a key role when the rice blast fungus is nitrogen starved,while the expression of MoGAP2 is not significantly different from the wild type,indicating that the permease may respond more specifically to oxidative stress.7.In the amino acid supplement experiment,we found that in the medium with aspartic acid,glutamate,glutamine or proline as sole nitrogen source,the growth rate of△MoGAP1 slows down and the mycelium grows thin,ΔMoGAP3 is affected in the media of aspartic acid and asparagine,whileΔMoGAP2 has no significant difference from the wild type.But when 1m M H₂O₂ is added to the media,ΔMoGAP2 cannot grow in the medium with aspartic acid as sole nitrogen source.The results indicates thatΔMoGAP2 loses the ability to utilize aspartic acid under oxidative stress.8.Through the p KD-7 complementary vector,we express a red fluorescent protein Ds Red2 at the C-terminus of MoGAP2,and then observe its subcellular localization using a fluorescence microscope.It is found that the fluorescence is mainly distributed in dots.After the mitochondrial fluorescence probe Mito-Tracker Green was used to specifically label the mycelial mitochondria,it was found that the red fluorescence and the green fluorescence had a more consistent distribution,indicating that MoGAP2 is most likely to be mainly distributed in mitochondria.The above results indicate that the general amino acid permeases MoGAP1,MoGAP2,and MoGAP3 are involved in the development and pathogenic process of M.oryzae while transporting amino acids.Especially in the initial stage of infection,MoGAPs have a stronger tolerance to the hydrogen peroxide stress of the host plant and the nitrogen starvation conditions in the biotroph stage.Our reaserch provides an important basis for elucidating the relevant mechanisms of nutrient absorption and metabolism pathway of M.oryzae in the future,and lays a theoretical foundation for the prevention and control of rice blast.