| Sugarcane (Saccharum officinarum) is the most important sugar crop in the worldwide, the sucrose accounted for92%of total sugar output in China. Plant disease is an important factor that affects the sugarcane yield and quality. Sugarcane smut (Sporisorium scitamineum) is one of the main diseases in the sugarcane planting area. It is generally believed that using sugarcane disease-resistant cultivar instead of susceptible one is the most economic and effective measure to prevent smut. At present, the related researches focus on the studies of cytology, morphology, physiology and biochemistry, as well as genetics, to explore the resistant mechanism to smut in sugarcane. While the research on the interaction between sugarcane and S. scitamineum at the molecular level still focuses on the cloning and quantification of a few resistance-related genes in the sugarcane genotypes after inoculation with S. scitamineum. These preliminary researches reflect the molecular mechanism of sugarcane resistant to S. scitamineum is regulated by multiple gene networks. But the above studies remain to be further. Using transcriptomics and proteomics to explore the impact of S. scitamineum on sugarcane can help to reveal the metabolic pathways, the molecular regulation networks and key genes during sugarcane response to S. scitamineum.In this study, a new detection method based on real-time fluorescent quantitative PCR (Q-PCR) was established, which could detect S. scitamineum rapidly and sensitively before disease symptom appear. It provides a scientific basis for rapidly detect and prevent S. scitamineum earlier. In this study, two sugarcane genotypes, one is an intergeneric BC2hybrid with good resistance characters named YachengO5-179from S. spp. hybrid X S. arundinaceum, the other is the present major cultivar "ROC"22which was susceptible to S. scitamineum, were taken as plant materials to establish affinity and the affinity interaction systems with S. scitamineum. The aim of this study was to reveal the molecular mechanism related to metabolism and signaling network of sugarcane response to S. scitamineum stress on the whole level through the RNA-Seq and iTRAQ technologies. Through multi-point time series comparison, the change of mRNA expression level was analysed. And the transcriptome expression profile was built in different time points in both sugarcane genotypes after inoculation with S. scitamineum. Then, three types of resistance-related genes, including P-l,3-glucanase, chitinase and catalase were mined, which can provide new gene resources for sugarcane breeding resistant to fungal diseases. The main results and conclusions are as follows.1. Smut is a fungal disease which widespread popularity in sugarcane planting area. Early detection and proper identification of S. scitamineum are essential in smut management practice. Here, we developed a TaqMan real-time PCR assay using specific primers (bEQ-F/bEQ-R) and a TaqMan probe (bEQ-P) which were designed based on the bE (b East mating-type) gene (GenBank Accession No. U61290.1). This method was more sensitive (a detection limit of10ag pbE DNA and0.8ng sugarcane genomic DNA) than that of conventional PCR (10fg and100ng, respectively). Reliability was demonstrated through the positive detection of samples collected from artificially-inoculated sugarcane plantlets (FN40). This assay was capable of detecting the smut pathogen at the initial stage (12h) of infection and suitable for inspection of sugarcane pathogen-free seed cane and seedlings. Furthermore, quantification of pathogen was verified in pathogen challenged buds in different sugarcane genotypes (Yacheng05-179and "ROC"22), which suggested its feasibility for evaluation of smut resistance in different sugarcane genotypes. Taken together, this novel assay can be used as a diagnostic tool for sensitive, accurate, fast and quantitative detection of the smut pathogen especially for asymptomatic seed cane or plants and evaluation of smut resistance of sugarcane genotypes.2. Samples of Yacheng05-179(resistance) and "ROC’22(susceptible) inoculated with sterile water for24h (control) and S. scitamineum for24h,48h and120h were used to analyze the transcriptome by Illumina RNA-Seq. The results showed that:(1) In total,36.68Gb data and181,603,016reads were generated from eight sugarcane samples through transcriptome sequencing. The total of148,605Unigenes were obtained by de novo assembly in separate samples, which contained46,525Unigenes at length of more than500bp and20,798Unigenes at length of more than1.0kb, while both Unigene were reduced to47,345and22,091after merging. Based on the Merge_assembly Unigene library, the gene structure was analyzed, including ORF prediction, simple sequence repeats (SSR) analysis and single nucleotide polymorphisms (SNP) analysis. Then,5,095SSR markers were generated. Comparing Unigene with NT, NR, SwissProt, TrEMBL, COG, GO and KEGG databases, bioinformatics annotation results of65,852Unigene were found. Besides,33,972Unigene (34.03%) had not been annotated. We speculate that these sequences without annotation may be new transcripts, which need further verification. The transcriptome database built in this study can provide reference for other related studies in sugarcane.(2) The analysis of gene expression quantity and the differential expression genes in each sample were performed, as well as the gene clustering pattern, functional annotation and enrichment analysis. The candidate genes were verified by Q-PCR method. The results suggest that:①The induction of the majority of the differential expression genes and metabolic pathways were common in the two sugarcane cultivars after challenge by S. scitamineum. Almost of them referred to all aspects of the life activities. These Unigene were involved in molecular functions, cellular components and biological process. The resistance related functional annotation was obtained, such as signal transduction mechanisms, energy production and conversion, inorganic ion transport, metabolism and defense mechanisms, etc. Pathway clustering analysis showed that, the differential expression genes involved in resistance related metabolic pathways of plant hormone signal transduction, flavonoid biosynthesis, plant-pathogen interaction and the cell wall fortification pathway, etc. Visibility, various of disease resistance pathways and the differential expression genes which referred to defense reaction and signal transduction were inspired in sugarcane after inoculation with S. scitamineum. These reveal that the molecular mechanism of response to smut pathogen infection was controlled by polygenic network system.②As the extension of inoculational time after S. scitamineum infection, the number of sugarcane differential expression genes were increased. What’s more, the total number of the up-regulated genes were significantly more than that of the down-regulated one. Although differential expression genes in YachengO5-179and "ROC"22were largely the same, the significant resistance related metabolic pathways were more than that of in "ROC"22. Meanwhile, the resistance related genes were expressed earlier (24h-48h) in YachengO5-179than that of in "ROC"22(48h-120h). And the gene expression pattern of up/down-regulation was more abundant in the resistant genotype. It reveals a resistance of specificity or earlier scheduling is performed in the affinity interaction. The potential function analysis of the above transcripts in sugarcane response to S. scitamineum can help us understand the role of these candidate genes in sugarcane response to smut pathogen infection, as well as in the study of the functional genomics of sugarcane disease resistance.3. On the basis of the transcriptome study, change of proteomics of YachengO5-179and "ROC"22inoculated with S. scitamineum after48h was analysed by iTRAQ technology. It showed that the number of identified proteins was4,251, including273differential expression proteins in YachengO5-179and341in "ROC"22. These differential expression proteins were significantly involved in phenylalanine metabolism, phenylpropanoid biosynthesis, biosynthesis of secondary metabolites, benzoxazinoid biosynthesis, fatty acid metabolism and biosynthesis of unsaturated fatty acids. The correlation analyses showed that after infection with S. scitamineum for48h, the correlations between the proteomics and transcriptomes were0.1502and0.2466in Yacheng05-179and "ROC"22, respectively. Twenty seven and ten differential expression proteins associated with the transcriptome were selected in the resistant variety and susceptible one, respectively. And the number of the differential expression proteins which were expressed at the same trend with differential expression genes were18and10, respectively. Most differential proteins were closely related to plant’s stress resistance, such as peroxidase, pathogenesis-related protein, beta-1,3-glucanase, heat shock protein and lectin. These results provide the basis for the determination of the subsequent metabolic pathways and the identification of key proteins.4. The transcripts of pathogenesis related proteins were differentially expressed after inoculated with S. scitamineum, involving10glucanase genes,26chitinase genes and1catalase gene. Studies on proteome associated with transcriptome showed pathogenesis-related protein1and beta-1,-3-glucanase are induced by S. scitamineum. As we know, pathogenesis related proteins are specific proteins induced by a sort of pathological conditions. They play an important role in the process of plant disease resistance reaction, and are closely related to systemic acquired resistance (SAR). According to the differential expression transcripts of these three kinds of typical pathogenesis related proteins, two β-1,3-glucanase genes ScGluAl and ScGluDl, one chitinase gene ScChi and one catalase gene ScCAT1were isolated from sugarcane by RT-PCR assay. The character of the spatio-temporal expression in differential expression genes was elucidated by Q-PCR. Subcellular location of fusion protein and the genetic transformation research of the target genes were applied by genetic engineering technology. This work will contribute to further genetic improvement of smut resistance in sugarcane by providing the excellent gene resources or technique.(1) β-1,3-glucanase can hydrolyze the main component of fungal cell wall of β-1,3-glucan, has been shown to express during plant-pathogen interaction and involved in plant defense response. In this study, β-1,3-glucanase enzyme activity in the resistant cultivar increased faster and lasted longer than that of the susceptible one when inoculated with S. scitamineum, along with a positive correlation between the activity of the β-1,3-glucanase and smut resistance. Furthermore, two β-1,3-glucanase genes from S. scitamineum infected sugarcane, ScGluA1(GenBank Accession No. KC848050) and ScGluD1(GenBank Accession No. KC848051), were cloned and characterized. Predicted amino acid sequences viz both ScGluAl and ScGluDl genes contain a glycosyl hydrolases family17domain, between which only29.25%consistency at the amino acid sequence level. Phylogenetic analysis suggested that ScGluAl and ScGluDl clustered within subfamily A and subfamily D, respectively. Agrobacterium-mediated transformation for transient expression of target genes (ScGluAl and ScGluDl) with green fluorescent protein (GFP) in onion epidermal peels demonstrated that both gene products were targeted to apoplast. Escherich coli Rosetta (DE3) cells expressing ScGluA1and ScGluD1showed varying degrees of tolerance to NaCl, CdCl2, PEG, CuCl2and ZnSO4·Q-PCR analysis showed up-regulation of ScGluA1and slight down-regulation of ScGluDl in response to S. scitamineum infection. It suggested that ScGluA1may be involved in the defense reaction of the sugarcane to the smut, while it is likely that ScGluD1was inhibited. Gene expression patterns of ScGluA1and ScGluDl, in response to other factors such as salicylic acid (SA), methyl jasmonate (MeJA), abscissic acid (ABA), NaCl, CdCl2and natural drought stresses, were similar to sugarcane response against smut infection. Over expression of ScGluA1in Nicotiana benthamiana showed an antimicrobial action on Fusarium solani var. coeruleum and Botrytis cinerea. Moreover, β-1,3-glucanase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. Together, β-1,3-glucanase may work as a component in sugarcane defense mechanisms for S. scitamineum. The positive responses of ScGluAl may contribute sugarcane resistance to adversity stress; while the negative responses of ScGluDl to biotic and abiotic stresses indicate it plays a different role from that of ScGluA1in interaction between sugarcane and biotic or abiotic stresses.(2) Chitinases (EC3.2.2.14), expressed during the plant-pathogen interaction, are associated with plant defense against pathogens. In the present study, a positive correlation between chitinase activity and sugarcane smut resistance was found. ScChi (GenBank Accession No. KF664180), a Class III chitinase gene encoded a31.37kDa polypeptide, was cloned and identified. Subcellular localization revealed ScChi targeting to the nucleus, cytoplasm and the plasma membrane. Q-PCR results showed that ScChi was highly expressed in leaf and stem epidermal tissues. The ScChi transcript was both higher and maintained longer in the resistance cultivar during challenge with S. scitamineum. The ScChi also showed obvious induction of transcription after treatment with SA, H2O2, MeJA, ABA, NaCl, CuCl2, PEG and low temperature (4℃). The expression levels of ScChi and six immunity associate marker genes were up-regulated by the transient over expression of ScChi. The histochemical assay of Nicotiana benthamiana leaves over-expressing pCAMBIA1301-ScChi exhibited deep DAB staining color and high conductivity, indicating the high level of H2O2accumulation. Besides, over-expressing ScChi helped improve N. benthamiana to defense Pseudomonas solanacearum, Fusarium solani var. coeruleum and Botrytis cinerea. Meanwhile, chitinase from To generation of ScChi transgenic N. benthamiana can inhibit the hypha growth of Fusarium solani var. coeruleum. These results suggest a close relationship between the expression of ScChi and plant immunity. In conclusion, the positive responses of ScChi to the biotic and abiotic stimuli reveal that this gene is a stress-related gene of sugarcane.(3) Catalase is an iron porphyrin enzyme, which serves as an efficient scavenger of reactive oxygen species (ROS) to avoid oxidative damage. In sugarcane, the enzymatic activity of catalase in the cultivar (Yacheng05-179) resistant to the smut pathogen S. scitamineum was always higher than that of the susceptible cultivar (Liucheng03-182), suggesting that catalase activity may have a positive correlation with smut resistance in sugarcane. To understand the function of catalase at the molecular level, a cDNA sequence of ScCAT1(GenBank Accession No. KF664183), was isolated from sugarcane infected by S. scitamineum. ScCAT1was predicted to encode492amino acid residues, and its deduced amino acid sequence shared a high degree of homology with other plant catalases. Enhanced growth of ScCAT1in recombinant Escherichia coli Rosetta cells under the stresses of CuCl2, CdCl2and NaCl indicated its high tolerance. Q-PCR results showed that ScCATl was expressed at relatively high levels in the bud, whereas expression was moderate in stem epidermis and stem pith. Different kinds of stresses, including S. scitamineum challenge, plant hormones (SA, MeJA and ABA) treatments, oxidative (H2O2) stress, heavy metal (CuCl2) and hyper-osmotic (PEG and NaC1) stresses, triggered a significant induction of ScCATl. The ScCAT1protein appeared to localize in plasma membrane and cytoplasm. Furthermore, histochemical assays using DAB and trypan blue staining, as well as conductivity measurement, indicated that ScCAT1may confer the sugarcane immunity. In conclusion, the positive response of ScCAT1to biotic and abiotic stresses suggests that ScCAT1is involved in protection of sugarcane against reactive oxidant-related environmental stimuli. |
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