| BLSB (Banded leaf and sheath blight), caused by Rhizoctonia solani Kiihn, is one of the most important diseases of maize (Zea mays. L) worldwide. Severe yield losses can result as a consequence of the rapid development and large-scale spread of the disease epidemic under optimal environmental conditions. Recently, yield losses approached 100% in southern of China when the ear rot phase predominated. Over the last few years, genetic and molecular studied on the disease and pathogen have been reported in maize. However, the molecular regulation mechanisms of disease-resistant involved in maize leaf sheaths infected by Banded leaf and sheath blight (BLSB) are poorly known. To gain insight into the transcriptome dynamics that are associated with their disease-resistant, based on phenotype traits of maize sheaths infection by R. solani and observation of maize sheaths infection by R. solani under scanning electron microscope, genome-wide gene expression profiling was conducted by Solexa sequencing.High-resistance maize inbred line seedlings of "R15" and high-sensitive maize inbred line seedlings of "Ye478" were treated with 7% hypochlorite solution for 30 min respectively, followed by three washes with sterilized water before being sowed in pots with autoclaved soil. Control plants were maintained under the same conditions. R. solani AG1-IA was cultured on potato dextrose agar (PDA) and incubated for three days at 28 ℃. Agar blocks (0.5 cm squares) were cut and prepared from the outer edge of a 3-day-old culture. The inoculated plants and mock-inoculated plants grew in the same growth chambers in Maize Research Institute of Sichuan Agricultural University. The leaf sheaths were covered with plastic bags to ensure high humidity.We observed phenotype traits of maize sheaths of high-resistance maize inbred line "R15" and high-sensitive maize inbred line "Ye478" infection by R. solani and observation of maize sheaths infection by R. solani under scanning electron microscope in R15. After that, high-resistance maize inbred line "R15" was screened as the sequencing materials and the inoculated and mock-inoculated bract tissues were collected at 0 h,6 h,12 h, and 24 h after 24h later inoculation for the four stages respectively. Two RNA libraries was constructed using RNA extracted and pooled in equal proportions from the leaf sheath of the BLSB resistant maize variety R15 at Oh,6 h,12 h, and 24 h after 24 h inoculation. Then sequenced using a Solexa sequencer at Huada Genomics Institute Co. Ltd, Shenzhen, China, three technical replicates with two biology replicates were used for each sample. Differential expression genes and their encoded transcription factors were identified, combing with BLSB-responsive differential expression miRNAs identified before, we constructed the putative regulatory network between miRNAs and transcription factors. We aimed to elucidate the biological functions of these DGEs, as well as transcription factors and miRNAs to provide deeper understanding in molecular mechanisms of sheath infected growth development and adversity resistance.1. Two RNA libraries was constructed using RNA extracted and pooled in equal proportions from the leaf sheath of the BLSB resistant maize variety R15 at 0 h,6 h,12 h, and 24 h after 24 h inoculation. Then sequenced using a Solexa sequencer at Huada Genomics Institute Co. Ltd, Shenzhen, China, three technical replicates with two biology replicates were used for each sample. More than four million tags were generated from sheath tissues without any leaf or development leaf, including 193,222 and 204,824 clean tags in the two libraries, respectively. Of these,82,864 (55.4%) and 91,678 (51.5%) tags were matched to the reference genes. The most differentially expressed tags with log2 ratio> 2 or<-2 (P< 0.001) were further analyzed, representing 1,476 up-regulated and 1,754 down-regulated genes, except for unknown transcripts. The expression patterns of candidate genes were assessed by quantitative real-time PCR, and it is showed the results were general agreement with the Solexa analysis, although the degree of change was lower in amplitude.2. GO Functional annotation of DEGs indicated that both up-regulated genes and down-regulated genes could be classified into 11 categories, such as catalytic, electrical carrier, transcription regulation and enzyme regulator. Significant GO analysis of DEGs in molecular function showed that the top one was transferase activity, transferring hexosyl groups, it is interesting that most of DEGs involved in biological process such as response to stimulus, multicellular organismal process and response to abiotic stimulus. These highly expressed genes were associated with defense, transport, transcription, signal transduction and metabolism, which were involved in senescence, abiotic and biotic stresses. In addition, KEGG pathway analysis showed that significantly differentially expressed genes enriched in metabolic pathways, plant-pathogen interaction pathways, plant hormone signal transduction, especially in starch and sucrose metabolic pathways, terpenoids and steroid synthesis, cysteine and methionine metabolism synthesis, In addition, a large number of differentially expressed genes involved in the ubiquitin regulation pathway and oxidative phosphorylation. Interestingly, a large number of differentially expressed genes were found involved in terpenoid and GA synthesis. And the candidate differentially expressed genes (DEGs) involved in terpenoid and GA synthesis were validated using qRT-PCR.3.156 transcription factors (TFs) were up-regulated and involved in maize sheath responsive to BLSB stress, including ERF transcription factors family (21), WRKY (18), bHLH (18), MYB (16), NAC (11), C2H2 (8), bZIP (6), GRAS (6), as well as ARF (2), TCP (2) and ZF-HD (2). And 245 transcription factors were also found down-regulated, such as ERF transcription factors family (18), WRKY (6), bHLH (24), MYB (17), NAC (8), C2H2 (14), bZIP (18), GRAS (10), ARF (8), HD-ZIP (10), SPL (5). Functional annotation of these transcription factors using Mapman found most of transcription factors involved in hormone signaling pathways and signal transduction pathway, as well as keeping redox state stability to take control of BLSB resistance.4. A comprehensive analysis of miRNAs selected from our previous deep sequencing results and associated target genes transcription factors under banded leaf and sheath blight (BLSB) stress caused by R. solani in maize. Eight differential expression miRNAs Zma-miR172e, Zma-miR160a, Zma-miR167c, Zma-miR171b, Zma-miR396c, Zma-miR159b, Zma-miR156k, Zma-miR319a association with TFs were selected from deep sequencing results and validated by qRT-PCR, together with their putative target genes transcription factors SBP、ARF、 GRAS、 AP2-ERF、MYB、TCP、GRF. The results showed that miRNAs were general agreement with deep sequencing results; transcription factors were also general agreement with Solexa analysis, except for TCP transcription factors. Moreover, majority of the analyzed miRNAs showed an inverse correlation with their corresponding predicted target genes transcription factors in this study.5. F-box mediated ubiquitin protein degradation pathway were involved in many biological processes in eukaryotes. The recent research showed that F-box TIR1 protein acted as auxin receptor. To explore the molecular mechanism between F-box TIR1 protein and its regulator Zma-miR393b, evolution of F-box TIR1 protein (GRMZM2G135978) were confirmed together with Arabidopsis F-box family and maize F-box family, and evolution of targeted miRNA393b were confirmed with other plant miRNA393 family. The results showed GRMZM2G135978 shared a branch with a large of Arabidopsis F-box family genes and F-box TIR1 protein present independent evolution and diversity. Zma-miR393b was highly conservative in miRNA393 family in different plants. We speculated that Zma-miR393b association with its putative target gene F-box TIR1 protein might function in biotic and abiotic stress, as well as plant development through F-box TIR1 mediated ubiquitin protein degradation pathway. We reported that Zma-miR393b down-regulates its putative target TIR1-like (F-box) gene by guiding the cleavage of their mRNAs in development of leaf sheaths in response to R. Solani infection, Zma-miR393b and its putative target gene TIR1 were confirmed through qRT-PCR and the spatial expression of Zma-miR393b was further analyzed by in situ hybridization. The results suggested that Zma-miR393b down-regulates F-box TIR1 genes in high-resistance maize inbred line R15 could respond to R. Solani infection earlier by accumulating more F-box TIR1 genes than high-sensitive maize inbred line Ye478. According to expression levers of the BLSB-responsive Zma-miR393b by in situ hybridization, it is demonstrated that leaf sheaths were infected by R. Solani spreading from outside to inside with the prolong of infection time and especially expressed in surrounding tissues of the primary xylem and metaxylem after 24 h.6. Based on the precious study and results obtained from our reseach, we constructed the miRNA-mRNA feedback regulatory pathway. It is demonstrated that plant can perception BLSB stress and a large number of BLSB association miRNAs were produced after pathogen attack (such as miR156, miR159 and miR160 et al), and then negative regulated their pupative target genes (most of them belong to transcription factors, including MYB, WRKY, SBP, ARF and NAC et al), thereby activating defense signal network to respond to pathogen invasion. In addition, R-genes would be produced from pattern recognition receptors (PRRs) that recognize conserved pathogenassociated molecular patterns (PAMP), R-proteins are mainly intracellular and have the capability to directly or indirectly detect isolate specific pathogen effectors encoded by avirulence genes (Avr) resulting in activation of defense specific signal transduction. |
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