Analysis Of Apoplastic Proteins And Environmental Factors Affecting The Maize-Fusarium Verticillioides Interaction

Maize(Zea mays L.)is one of the most important food crops and provides raw materials for industrial uses.The sound development of the maize industry has a strategic significance for the food security and stable supply of poultry and livestock products.Maize ear and stalk rot caused by Fusarium verticillioides is one of the major diseases that threaten the development of the maize industry.Understanding the influence of environmental conditions and pathogen processes provides a theoretical basis for preventing the disease.In this study,we have analyzed the interaction of F.verticillioides with maize through apoplastic secretory proteins.Environmental factors including temperature and water activity have also been studied for the growth,mycotoxin production and transcriptome of F.verticillioides.The study points out that:1.During the attack of a pathogen,various defence-associated proteins are released by the host plant in the apoplast to impede the perceived attack.We have utilized mass spectrometry(LC-MS/MS)and label-free quantification to analyze the apoplastic washing fluid(AWF)from maize stalk and identified the proteins responsive to the Fusarium verticillioides infection.We have identified 742 proteins,and among these,119 proteins were differentially accumulated(DAPs),i.e.,35 up-regulated,18 downregulated,and 66 proteins were only induced by the pathogen infection.The differentially accumulated proteins were analyzed for their Gene Ontology(GO)and Kyoto Encyclopedia of Gene and Genomes(KEGG)pathway enrichment.The highly enriched Biological Process(BP)term was the L-serine biosynthesis process,whereas the most enriched Molecular Function(MF)term was the cysteine-type endopeptidase inhibitor activity.It was also found that the pathways related to the biosynthesis of amino acid,biosynthesis of secondary metabolites,protein processing in the endoplasmic reticulum,and carbohydrate metabolic pathways were significantly enriched.Moreover,61 out of 119 differentially accumulated proteins were predicted as secretory proteins.The secretory pathways analysis showed that a greater number of proteins were secreted through the conventional secretion system compared to the unconventional secretion system.The identified secreted proteins were related to a variety of pathways in defence responses,including cell redox homeostasis,cell wall modification,signal transduction,carbohydrate metabolism,binding proteins(metal ion binding,RNA binding and,heme-binding),maintenance and stabilization of other proteins,indicating a complex response from the plant to the fungal infection.Our data suggested that a number of host proteins belonging to various pathways have been modulated in the apoplastic region after F.verticillioides infection.2.The proteomic analysis of the apoplastic washing fluid(AWF)of maize identified 50 apoplastic effector protein from Fusarium verticillioides.Out of them,36 proteins showed their ability to suppress cell death induced by other cell death-inducing proteins.AP20 was randomly selected for further characterization.The phylogenetic analysis confirmed that AP20 is conserved in Fusarium genera and other pathogenic fungal organisms.Various databases,including Uniprot,Wolfpsort and Signal P,predicted AP20 as a secretory protein and the presence of signal peptide was predicted in the N-terminal of the AP20.Notably,AP20 is an uncharacterized protein,and no study has been published on this protein.We verified the signal peptide activity using a yeast invertase secretion system to confirm the secretory nature of AP20.The subcellular localization studies of AP20 confirmed that AP20 has localized in the cytoplasm and the nucleus.The innate immune response,including the ROS burst and callose deposition,initiated by flg22 treatment,was suppressed by AP20.Moreover,the expression of PTI marker genes was also suppressed by the protein.The pathogenicity of the deletion mutant was reduced to the maize seeds and seedlings.These results indicated that AP20 is a secreted protein that negatively affects plant immunity and is necessary for the growth of the pathogen in-planta.3.The effect of water activity and temperature on the growth,Fumonisins B1 production and transcriptome of F.verticillioides was studied by inoculating the maize kernels having different aw(0.98,0.93)and cultured at 15 ℃ and 25 ℃.The optimal growth conditions of F.verticillioides were aw 0.98,and 25 ℃ and FB1 were highest at aw 0.98 and 15 ℃.The transcriptome analysis showed that the differentially expressed genes(DEGs)caused by temperature were higher in number than water activity.Gene Ontology(GO)enrichment analysis of temperature-induced DEGs revealed that the up-regulated genes were mainly involved in the macromolecular complexes.At the same time,the down-regulated genes were mainly involved in the different enzyme activity.In contrast,for water activity,the genes were mainly involved in carbohydrate metabolic processes.Temperature and water activity also influences the genes responsible for the fumonisin production,like FUM gene cluster,the genes responsible for fumonisin but resides outside the FUM cluster and the genes responsible for secondary metabolite production.It can be concluded that during the interaction of F.verticillioides with maize,a tremendous proteomic reprogramming is done by the host plant in the apoplast to deter the invading pathogen and the pathogen also uses its protein weaponry to manipulate the host immunity to establish a pathogenic relationship.