| Sugarcane(Saccharum spp.L.)is a perennial gramineous plant crop cultivated in tropical and subtropical regions.It is the largest sugar crop in the world.Ratoon stunting disease(RSD)caused by the Gram-positive bacterium Leifsonia xyli subsp.xyli(Lxx)is common in sugarcane planting areas,seriously affecting the growth of sugarcane and resulting in yield and economic loss.Previous studies have mainly focused on the occurrence and detection of RSD,the isolation and culture of Lxx,and the ultrastructure observation on sugarcane in response to RSD stress.The molecular mechanism of sugarcane response to the infection of Lxx is still limited to the cloning and quantitative expression of the differentially expressed genes of sugarcane under RSD stress.Since it is difficult to isolate and culture Lxx in vitro and carry out genetic manipulation on it,the study on the interaction mechanism between Lxx and sugarcane remains limited.In this study,the effects of Lxx infection on the growth,and physiological and biochemical metabolisms of sugarcane were observed after inoculation.The interactions between sugarcane and Lxx was investigated under RSD stress on Badila,a highly RSD susceptible cultivar of Saccharum officinarum.The changes of m RNA and protein expression levels in different parts of sugarcane plant in response to the stress of Lxx were analyzed by using high-throughput RNA-Seq and DIA technology.The phosphoproteomics in sugarcane infected with Lxx were analyzed by i TRAQ technique.In addition,the function of the Lxx membrane protein gene Lxx18460(anti-sigma K)was preliminarily analyzed by gene transformation.The Lxx18460 gene was cloned,the plant expression vector was constructed,and the genetic transformation was carried out by agrobacterium-mediated method.The main results were as follows.1.Healthy seedcanes of the sugarcane cultivars Badila and GT11 were used as experimental materials for Lxx inoculation treatment.Lxx18460,a specific gene of pathogenic bacterium Lxx,was analyzed at 50 d,180 d and 210 d after Lxx inoculation in sugarcane by means of real-time quantitative PCR and Western Blot.The results showed that,the Lxx18460 expression level gradually increased with the growth of sugarcane after inoculation of Lxx.After Lxx infection,the plant height,stalk diameter,single stalk weight,water potential,and contents of cysteine and methionine decreased,while the membrane permeability,and contents of free amino acid and calmodulin increased.At the same time,the relative expressions of resistant genes PAL,ZFP and NBS-LRR were also up-regulated after Lxx infection in sugarcane.2.Disease-free cane setts of the sugarcane cultivars Badila and GT11 were inoculated with Lxx.The analyses at four stages showed that the contents of cellulose,hemicellulose and pectin decreased by 14.88%,13.74% and 11.17% under Lxx infection,respectively,while the contents of callose and lignin increased by 13.13% and 15.29%,respectively,in the Lxx infected leaves of Badila.In GT11,the contents of cellulose,hemicellulose and pectin decreased by 7.43%,5.43% and 16.7%,respectively,while the contents of callose and lignin increased by 17.45% and 15.07%,respectively,in the Lxx infected leaves.After infected by Lxx,the total dry matter accumulation,total nitrogen,total phosphorus and total potassium of Badila decreased by 53.5%,12.8%,11.7% and 7.4%,respectively,while those of GT11 decreased by 21.8%,33.8%,6.5% and 5.7%,respectively.There were significant differences between the two varieties.3.Illumina RNA-Seq was performed for the leaves and base stalks of sugarcane after 90 days of Lxx inoculation.There were 11,802 differentially expressed genes(DEGs)in the RSD infected leaves compared with healthy leaves,among which 7,721 were up-regulated and4,081 were down-regulated.There were 9,325 DEGs in the infected sugarcane stalks,among which 4,426 were up-regulated and 5,059 down-regulated.These DEGs have different biological functions and involve in different metabolic pathways in leaves and stalks.In summary,sugarcane responded to RSD stress in the signaling pathway of photosynthesis,plant and pathogen interaction,plant hormone and secondary metabolism,and cell wall related pathways.4.Based on transcriptomic analyses,DIA technique was used to analyze the proteomics.The results showed that the number of proteins identified was 9,702 from 12 sugarcane samples.There were 98 differentially expressed proteins(DEPs)in the Lxx-infected leaves compared with healthy leaves,among which 40 were up-regulated and 58 down-regulated.There were 407 DEPs in the Lxx-infected sugarcane stalks,among which 179 and 228 were up-regulated and down-regulated,respectively.The results showed that sugarcane stems responded more actively and sensitively to the infection of RSD than leaves.The DEPs were significantly enriched in plant hormone signal transduction,phenylpropyl and glutathione biosynthesis,plant and pathogen interaction pathways,etc.These metabolic pathways were directly or indirectly involved in the response of sugarcane to RSD.The correlations between the DEPs and the corresponding DEGs were 0.1545 in leaves and 0.3076 in stalks.Eleven and thirty eight DEPs were found associated with the transcriptomic genes in the leaves and stalks,respectively.And the DEPs which were expressed in the same trend with the differential expression genes were 7 and 27,respectively.Most of the associated proteins were involved in plant hormone signal transduction,plant pathogen interactions,and plant secondary metabolites synthesis,etc.5.The phosphorylated proteins in Badila infected with Lxx were studied by i TRAQ technique.The results showed that a total of 3,924 phosphorylated peptides were identified in6 sugarcane samples,and 3,326 phosphorylated sites were located in 1,879 phosphorylated proteins.After 90 days of Lxx inoculation,there were 114 differential phosphorylated peptides in the Lxx infected sugarcane stalks compared with the healthy control plants,among which63 were up-regulated and 51 down-regulated.The enrichment analysis of the pathways of differential phosphorylated proteins showed that more differential phosphorylated proteins were involved in the metabolic pathway,the synthesis of secondary metabolites and the interaction between plant pathogens.Metabolism of alanine,aspartic acid and glutamate,and the biosynthesis of arginine were the significantly enriched pathways.6.The membrane protein gene Lxx18460(anti-sigma K)of Lxx was transformed into tobacco.Compared with the wild type,the Lxx18460-transgenic tobacco plants had lower plant height,smaller leaf area and lower biomass.It was found that the net photosynthetic rate of the transgenic plants was reduced and the endogenous hormone synthesis was inhibited.Lxx18460 is mainly highly expressed in tobacco stems.During the plant growth process,the expression of Lxx18460 at the transcription and translation levels increased steadily with the growth of tobacco,and its expression slowed down the growth of tobacco plants.The higher the expression of Lxx18460,the more obvious the influence on tobacco.A monocotyledon plant expression vector p UBTC-Lxx18460 controlled by the Ubi promoter was constructed successfully.Sugarcane callus was infected by agrobacterium-mediated transformation.By this way,the vector was introduced into the sugarcane cultivar ROC22.The transgenic sugarcane plants were confirmed by PCR and Western Blot.The results of target protein detection also indicated that the gene was correctly expressed as the corresponding protein in the transformed plants.Overall,this study investigated the physiological and molecular mechanisms of sugarcane in response to RSD by analyzing the physiological and pathological changes in sugarcane caused by Lxx infection,and preliminarily investigated the functions of the gene Lxx18460.These work will enable us to have a deeper understanding of the response mechanism of sugarcane to RSD and provide a reference for further studying the interaction between RSD and sugarcane. |
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