Molecular Mechanism Of Aspergillus Flavus Infecting Rice Based On Transcriptomics And Metabolomics

Aspergillus flavus(A.flavus),one of the dominant molds in rice,will lurk on the surface of healthy grains,causing mildew in rice and affecting grain quality.Rice in China is generally contaminated by aflatoxins(AFs)produced by A.flavus.A.flavus has seriously threatened food safety and human health,restricting the development of rice industry and people's needs for better life.At present,the research on mold contamination is mainly focused on detoxification of grains after harvest,and there are few studies on the infection process and mechanism of pathogens on rice.In this paper,molecular mechanism of A.flavus infecting rice was initially clarified by apparent observation,total number of molds,AFB₁ content of infected rice,transcriptomics,physical and chemical indicators,and metabolomics analysis of A.flavus.Molecular mechanism of A.flavus infecting rice provided a theoretical basis for the prevention and control of post-harvest grain infection by pathogens and green preservation technology.The research results were as follows:(1)The process of A.flavus infecting rice was obtained by observing morphological changes of A.flavus on rice,and combining the effects on infected rice appearance,total mold and AFB₁content.The growth process of A.flavus on rice was explored,and the infection period and key time point were determined.Results showed that A.flavus could be activated under suitable conditions(Moisture content of rice:27.5±0.5%(dry basis);Temperature:29±1?;Humidity:85±1%;Dark),and mycelium would appear on rice husk within 3 d.After 3 d,the infection of A.flavus on surface of rice husks intensified day by day,and infected rice began to mildew and produce toxins on the 3^rd day,until the 7^th day was severely mildewed and had the highest toxicity.It also had an impact on the color of rice.Therefore,the first 3 days were selected as the pre-infection period,the last 4 days as the post-infection period,and the 3^rd day was selected as the key time point of infection.The part of mycelium that grown on rice husks infected interior of grain in two ways:one was to directly infect by penetrating rice husks,and the other was to obtain the nutrients necessary for its growth from the wound when it is difficult to penetrate rice husks.A.flavus infected on cracks in grain husks.(2)A.flavus on PDA medium was taken as a blank,high-throughput transcriptome sequencing of A.flavus on rice in the pre-infection period(0?3 d)and post-infection period(4?7 d)was performed.Bioinformatics analysis was performed.Differentially expressed genes and related metabolic pathways provided some molecular insights for mechanism of A.flavus infection on harvest of fresh rice.Changes of A.flavus protein content,ATP concentration,ion ATPase activities and other physical and chemical indicators provided verification for the transcriptome data and conclusions during the infection process.It was found that A.flavus cultured on PDA and rice on the 3^rd day,a total of 5283 differentially expressed genes were found,of which 3313 genes were up-regulated and 1970 genes were down-regulated.A.flavus cultured on the 7^th d had a total of 5,074 differences expressed genes,of which 2553 genes were up-regulated and2474 genes were down-regulated.Key differentially expressed genes mainly involved cell wall degrading enzyme,amino acid metabolism,energy regulation and toxin metabolism.(3)Using ultra high-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry(UPLC-Q-TOF-MS)technology to detect the metabolites of A.flavus.And combining with the analysis of multivariate statistical variables to screen out differentially expressed metabolites.Changes of metabolites and main metabolic pathways involved in A.flavus in the process of infecting rice were analyzed.There were 537 differentially expressed metabolites through PCA and OPLS-DA analysis.Volcano maps showed that there were 65 differentially expressed metabolites in the pre-infection period,of which 45 were up-regulated and 20 were down-regulated.There was a total of 138 differentially expressed metabolites,of which 132 were up-regulated and 6 were down-regulated in the post-infection period.Key differentially expressed metabolites mainly involved amino acid metabolism,fatty acid biosynthesis,secondary metabolite synthesis and other pathways.Based on the above research,it could be found that A.flavus can be activated and begin to colonize after inoculation on rice.The infection process was divided into the pre-infection period and the post-infection period.The specific infection process and mechanism were as follows:A.flavus that infected rice first degraded polysaccharides such as cellulose on the surface of rice.The down-regulated expression gene cre A promoted the expression of cellulase and enabled cells to quickly utilize polysaccharides.The metabolite oleic acid was used as a nutrient carbon source and energy source for A.flavus to promote infection on the surface of rice.The entry of acetate unit into the toxin synthesis pathway was inhibited.While afl H,hyp D,and hyp B were significantly up-regulated to stimulate the toxin synthesis and start to produce toxins to complete the pre-infection(infection of the rice surface).In post-infection period,the metabolite acetyl-N-formyl-5-methoxykynurenine was produced,and A.flavus was affected by the defense mechanism of rice(host).Part of the toxin-producing regulatory genes stimulated the synthesis of secondary metabolites such as toxins,especially the up-regulation of afl S accelerated toxin synthesis rate,and finally caused toxin production of A.flavus on rice to be higher than that in the medium substrate to complete the internal infection of rice.At the same time,the up-regulation of vitamin B₆,alanine involved in amino acid metabolism,and galactose-1-phosphate involved in energy metabolism ensure the production of acetyl-Co A and the energy supply of A.flavus infecting rice tissues.Genes related to glyceride metabolism pathway were significantly up-regulated,and stearic acid involved in fatty acid biosynthesis in metabolites was up-regulated to ensure the production of glycerol and the normal supply of fatty acids.Glycerides and fatty acids played an important role in maintaining and regulating the normal biological functions of cells,ensuring the functional integrity of A.flavus,and promoting the continuous infection of A.flavus.