| Sugarcane smut is a fungal disease caused by Sporisorium scitamineum. Cultivation and promotion of sugarcane cultivar with smut resistance is the most efficient way to control this disease. Regarding smut resistance, many researches from the aspects of morphology, cytology, and physiological and biochemical, and molecular interaction mechanisam have been carried out. They demonstrated that the interactions between sugarcane and S. scitamineum was a complex network. The present study aimed to reveal the responses of sugarcane to S. scitamineum from multiple orientations and more angles. Firstly, the proliferation of S. scitamineum in the bud tissue and the activity of key enzyme from reactive oxygen metabolism and phenylpropanoid pathway were analyzed to obtain critical times of interactions between sugarcane and S. scitamineum, and also thus accurately understood the response time and strength of sugarcane to S. scitamineum infection. Secondly, target genes spliced by miRNA and their degradation site were identified using degradome sequencing technology. Thirdly, combined differential expression and functional classification of target genes were also conducted to explore disease-related target genes. Finally, qRT-PCR was performed to analyze the expression of the differentially expressed target genes and their corresponding miRNA, thus the spliced role of corresponding miRNA could be further characterized. Besides, the metabolic pathways of target genes were also analysed to further understand the response mechanism of sugarcane to the infection by S. scitamineum, which should provide gene and miRNA resources for the genetic improvement of sugarcane smut resistance. The main results are as follows:1. YC05-179 (resistance) and ROC22 (susceptible) act as the research materials. Sugarcane buds inoculated with S. scitamineum (treatment group) and sterile water (control group) were selected after inoculated 0 d,1 d,2 d, 3 d,5 d and 7 d. The situation of S. scitamineum proliferation was detected using qRT-PCR method. Activity of the key enzymes was measured from reactive oxygen metabolism (POD, SOD and CAT) and phenylpropanoid metabolism pathway (PPO, PAL and TAL). After inoculation with S. scitamineum, the amount of S. scitamineum increased with the extension of inoculation time in both YC05-179 and ROC22, while the amount of S. scitamineum in ROC22 was more than that in YC05-179 at the corresponding time point. After inoculated by S. scitamineum, POD, SOD and CAT activity in YC05-179 was generally higher than that in the control group. The activity of POD and CAT in YC05-179 was also significantly higher than that in ROC22 at the corresponding time point, and their peaks also appeared earlier. Besides, results also showed that the strength of the active oxygen metabolism pathway in YC05-179 was higher than that in ROC22 at the corresponding time point, resulting in its earlier response to the infection of S. scitamineum. At the same time, the metabolic pathway catalyzed by TAL acted as main entrance of phenylpropanoid metabolism pathway after inoculated by S. scitamineum, while the phenylpropanoid metabolism pathway was weak. Along with the extension of inoculation time, PAL and TAL worked together to enhance the phenylpropanoid metabolism pathway, and the pathway catalyzed by PAL can also be launched faster in YC05-179. The reactive oxygen metabolism and phenylpropanoid metabolism pathway in buds tissues were all stronger after inoculated 5 d by S. scitamineum, followed by 2 d and 3 d. Combined with the situation that the obvious difference of the amount of S. scitamineum appeared in two sugarcane cultivars after inoculated 2 d by S. scitamineum, it was speculated that 2 d and 5 d were the best time points to collect the samples for the research on degradome sequencing of target genes during the interaction between sugarcane and S. scitamineum.2. YC05-179 and ROC22 acted as the research materials. The sugarcane buds inoculated with S. scitamineum for 2 d and 5 d were used to conduct degradome sequencing array, and the buds with sterile water for 0 d acted as control material. There was 122.33 M amount of raw data in 6 samples. Q30 is over 93% each sample after data was evaluated. The sequences for analysing degradation site could perfectly match to reference sequence of sugarcane. The results showed that the quality of degradome sequencing was high. In total,309 target mRNAs were detected for 337 degradation sites in 6 sugarcane samples, corresponding to 97 known miRNAs and 112 novel miRNAs, which showed that target genes can be cleaved by miRNAs at several different sites. Target gene was given priority to Category 0, involving a variety of regulation process of life, such as signal transduction mechanism, inorganic ion transport and metabolism, energy production and conversion, defense mechanism, translation, posttranslational modification, energy production and conversion and glycerolipid metabolism, etc. GO analysis indicated that target classification of differentially expressed target genes in two sugarcane cultivars involved in cellular component containing cells, membrane, arganelle and cell part and other cell components. They mainly played catalytic activity and binding function, participated in the metabolic process, cellular process, single-organism process, responsed to stimulus, biological regulation, immune system process and signaling pathways, etc. KEGG enrichment analysis showed that differentially expressed target genes involved in metabolic pathways associated with smut resistance, for example, plant hormone signal transduction, ubiquinone and other terpenoid-quinone biosynthesis, ubiquitin mediated proteolysis, plant-pathogen interaction, oxidative phosphorylation, peroxisome, phenylalanine, tyrosine and tryptophan biosynthesis, phagosome, etc.3. qRT-PCR analysis was performed for the differentially expressed target genes and their corresponding miRNA Results showed that target genes and their corresponding miRNA had opposite expression patterns in at least one sugarcane cultivar. This expression pattern was in line with splied action for miRNA CCR involved in lignin biosynthesis pathway. After inoculated by S. scitamineum, the increased expression of several genes/miRNA contributed to the biosynthesis and accumulation of lignin, which responsed to the infection of S. scitamineum. UCH-L5 was closely related to ubiquitin mediated proteolysis, which involved in the response of sugarcane to S. scitamineum infection. GK, MLO and HIR1 involved in plant-pathogen interaction. Among them, GK possessed NHO1 activity, and its increased expression could enhance sugarcane resistance to S. scitamineum. MLO involved in calcium ion pathway, and its activity had a negative correlation with sugarcane resistance to S. scitamineum. After inoculated by S. scitamineum, MLO gene expression declined, thus for reduced the inhibition effect of MLO protein to defense response. MR participated in hypersensitive response in plants. After inoculated by S. scitamineum, the expression of HIR1 rose, thus buds tissue could induce early hypersensitive response to limit the further expansion of S. scitamineum. EIL3 was transcription factors of ethylene signaling pathways. After inoculated by S. scitamineum, the increased expression of EIL3 enhanced ethylene signaling pathway, availing to resist the infection of S. scitamineum. ARF and AIP involved in auxin signaling pathways. After inoculated by S. scitamineum, ARF8 was down-regulation, the descend range of ARF8 was lesser. Simultaneously, AIP was up-regulated, and the ascent range of AIP was larger in YC05-179. These results displayed that auxin signaling pathways can reply to infection of S. scitamineum. In addition, the lower expression of GRF8 illustrated that the growth of sugarcane was inhibited to some extent. SAMDC participated in polyamine biosynthesis. After inoculated by S. scitamineum, SAMDC was up-regulated, causing polyamine metabolic pathways enhancement. MYB was a typical resistance-related transcription factor. The expression trend and response time of MYB2 was different in two sugarcane cultivars, suggesting the association of response ability of MYB2 with the smut resistance levels of sugarcane genotypes. AGO IB had opposite expression patterns in two sugarcane cultivars. In YC05-179, AGO 1B was down-regulatied, availing to more accumulation of resistance-related target genes which could be cleaved by miRNA This showed that miRNA feedback regulation may also participate in the response of sugarcane to S. scitamineum. However, since the expression patterns of AGO 1B, PP2C 6, HIR1, AIP and SAMDC had difference with the results of degradation sequencing, the roles of these genes/miRNA in the response of sugarcane to S. scitamineum were needed to further validated. |
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