| The common bunt is a seedborne disease in many areas of the world.The primary pathogen that cause this disease is the fungus Tilletia laevis Kühn,is the obligate biotroph that seriously reduces wheat grain quality and yield in some regions of the world.The disease caused serious harm to the safety production of wheat and resulted in serious agricultural economic losses.The pathogen cause a systemic infection initiated by root infection followed by growth through the wheat plant to reach the immature inflorescence meristems under favorable condition.The pathogens cause mild or no symptoms in the vegetative organs,but as they reach in anthers and ovaries,they grow vigorously and disrupt the normal structure of both.At present,there are few studies on the effects of Tilletia laevis infection on the development of wheat flarol organ,especially the studies on anthers.For proven Tilletia laevis on wheat anther development,using Tilletia laevis and susceptible cultivars “Dongxuan 3” as the experimental material,phenotypic observations of plants,anthers,tapetum and pollen were carried out by using microscopic techniques at different developmental stages of anther.The transcriptome sequencing analysis was conducted in anthers at the seven developmental stages,and the results were further verified by physiological index determination and quantitative analysis.At the same time,in order to solve the problems of low germination rate of teliospores and low infection rate of wheat plants under artificial culture conditions,the liquid culture medium of Tilletia laevis was developed and the artificial culture conditions were optimized.In order to qualitatively and quantitatively study the extracellular metabolites and their effects on development of Tilletia laevis,we used Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer(UPLC-MS)to conduct a wide range of targeted metabolomics studies on fungus liquid at different developmental stages.Main contributions of this work are as follows:1.To soil extract p H,table speed,inoculation amount for single factor,analyses the three factors on the Tilletia laevis teliospores germination rate,the influence of the orthogonal experiment on the three factors,combined with the team to determine the Tilletia laevis teliospores germination of optimum temperature,optimizing selected teliospores germination of liquid culture conditions for 16 ℃,p H 7.0,speed 150 RPM,quantity of 1.2 x 105 spores/ml.The germination rate of teliospores and infection rate of wheat plant were increased by 10% and 5% by soil extract method.2.We used Ultra High Performance Liquid Chromatography Q Exactive Mass Spectrometer(UPLC-MS)to conduct a wide range of targeted metabolomics studies on fungus liquid at five different developmental stages: teliospore,promycelium,primary basidiospore,H-body,and secondary basidiospore.A total of 743 metabolites were detected from the Tilletia laevis suspension,101 of them significantly differential between stages.These were mainly organic acids and their derivatives,organoheterocyclic compounds,lipids,and lipid-like molecules.Analysis of the KEGG metabolic pathways showed that the differential metabolites were significantly enriched in 15 metabolic pathways,including protein digestion and absorption,biosynthesis of amino acids,and central carbohydrate metabolism in cancer and so on.This study provides a theoretical basis for the in-depth study of the extracellular active substances of Tilletia laevis and provides a reference for their analysis.In the comparison between CK and mycelium at different developmental stages,there were 285 differential metabolites in the T1 vs CK comparison.In the comparison of mycelium at consecutive developmental stages,there were 112 differential metabolites in the T1 vs T2 comparison.There were 77 differential metabolites in T2 vs T3.There were 40 differential metabolites in T3 vs T4.There were 84 differential metabolites in T4 vs T5.3.In this study,we ascertained the timeline of anther development in wheat first time using confocal microscopy.The timeline of anther development can be divided into seven stages: before meiosis stage,meiosis stage,tetrad stage,earlynucleate stage,latenucleate stage,binucleate pollen stage,trinucleate pollen stage.Our results showed that hyphae of Tilletia laevis was well established on epidermis cells at 1020 μm long and rupture the cell structure.Epidermis cells are the most earliest infected anther cells from filaments,from which the hyphae spread to other connective cells.To understand the infection between somatic and reproductive cell in wheat anther,cell number and size were estimated in infected anthers.In present experiment,we analyzed the effect the Tilletia laevis on anther development to address the question of whether the pathogen cause alterations in cell count and sizes after successful infection.Our results showed that cell count of epidermis cells,endothecium cells and middle layer cells significantly decreased,but no statistically observation was noted in the cell sizes of epidermis cells,endothecium cells,and middle layer cells after Tilletia laevis infection.Infection caused by delayed disintegration of the middle layer cells and tapetum,tapetum by monocytes into dual-core and by dual-core cells into glandular cells are a phase delay,tapetum apoptosis abnormal changes caused by unequal division of pollen mother cell,produce micronucleus,tetrad callose delay degradation,after small spores cannot develop into mature pollen.In order to protect the plant,the activities of SOD,CAT and POD enzymes were significantly up-regulated.These results suggested that the critical stage of pollen abortion was latenucleate and tapetum programmed cell death disorder was the main cause of pollen abortion.4.High quality clean data of 495.08 GB were obtained by transcriptome sequencing analysis of healthy anther and Tilletia laevis infected anther at the seven developmental stages.Contrasted with healthy anther 27164 differentially expressed genes(DEGs)were obtained from Tilletia laevis infected anther,which included 10649 up-regulated genes and16515 down-regulated genes.There were 3553 up-regulated genes and 2083down-regulated genes in before meiosis stage,930 up-regulated genes and 4070down-regulated genes in meiosis stage,186 up-regulated genes and 1056 down-regulated genes in tetrad stage,960 up-regulated genes and 1811 down-regulated genes in earlynucleate stage,2267 up-regulated genes and 2627 down-regulated genes in latenucleate stage,1129 up-regulated genes and 2286 down-regulated genes in binucleate pollen stage,1624 up-regulated genes and 2582 down-regulated genes in meiosis stage.The distribution of up-regulated and down-regulated genes showed that with the prolonging of Tilletia laevis infected anther development,the down-regulated genes were significantly more than the up-regulated ones.Through KEGG pathway analysis of the differentially expressed genes,it was found that the five most concentrated pathways were: Protein processing in endoplasmic reticulum,Starch and sucrose metabolism,Plant-pathogen interaction,Phenylpropanoid biosynthesis and Plant hormone signal transduction.The results of physiological indices,histochemical analysis and quantitative analysis showed that the decreased starch and sucrose contents were appeared due to the influence of down-regulated genes in these pathways,the activities of cell wall invertase and vacuolar invertase in anthers were significantly decreased.Based on the above results,the sucrose transport of tapetum and microspore in the early anther development was disturbed,starch synthesis was blocked,and nutrient supply of microspore was insufficient,which eventually led to pollen abortion.The mechanism of anther abortion induced by wheat glabra is closely related to the obstruction of sugar transportation. |
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