Studies On The Influencing Factors And Related Genes Of Melanin Formation In Rhizoctonia Solani AG-1 IA

Rice sheath blight,caused by Rhizoctonia solani AG-1 IA,is one of the most important fungal diseases of rice worldwide and causes severe yield losses in rice production.R.solani AG-1 IA survives in soil with its predominant state of mycelia or sclerotia,and melanization of fungi may contribute to enhance the virulence and resistance of fungi to diverse environments.In order to elucidate the molecular mechanism of melanin formation and its function during the pathogenesis of R.solani AG-1 IA,a series of studies on the melanin of R.solani AG-1 IA have been carried out.The main research results were reported as follows:1.Four kinds of chemical substances,i.e.chemical fertilizer group?500?g/mL of K₂SO₄,NaH₂PO₄,CO?NH₂?₂ solution?,metal ion compound group?5?g/mL of CuSO₄,FeSO₄,ZnSO₄ solution?,antioxidant group?5?g/mL of quercetin,morin,vitamin C solution?and contrast group?300?g/mL of hyoscyamine,50?g/mL catechol?,were evaluated for their effects on the mycelial growth rate,sclerotial number,distribution situation,fresh weight and dry weight as well as the content of melanin of R.solani AG-1IA,and the relationship between these substances and the mycelial growth,sclerotial development and melanin formation would be further calarified so as to finally elucidate the effects of chemical substances on the melanin formation of R.solani AG-1 IA.The results showed that the same concentration and different chemical substances had different effects on mycelial growth rate,sclerotial number,distribution situation,fresh weight and dry weight as well as the content of melanin;there was no interrelated relation among melanin formation,mycelial growth and sclerotial development,i.e.the chemical substances,which have inhibitory action on mycelial and sclerotia formation,might have no inhibition on melanin formation.The melanin was observed by UV visible spectrum,Fourier infrared spectrum and liquid chromatography-mass spectrometry?LC-MS?to confirm that the melanin of R.solani was catechol melanin.2.Amino acid compositions in the R.solani AG-1 IA mycelia and fermentation liquid were analysised.The results showed that the contents of 3 kinds of amino acids,i.e.aspartic acid,glutamic acid and alanine,in the mycelia of R.solani AG-1 IA were relatively rich,among them,the content of glutamic acid was the highest;in the fermentation liquid of R.solani AG-1 IA,the contents of aspartic acid and glutamic acid were the most abundant in the mycelium or fermented liquid of R.solani AG-1 IA.The content of precursor tyrosine of melanin formation in mycelia was no significant difference in the comparison of the three mycelia of R.solani AG-1 IA;however,the content of precursor phenylalanine of melanin formation in 300?g/mL hyoscyamine treatment were significant differences with the comparison of control and catechol treatment,while the control and50?g/mL catechol treatment had no significant difference.The tyrosine of control of50?g/mL catechol treatment and control of R.solani AG-1 IA in fermentation liquid had no significant differences,but 300?g/mL hyoscyamine treatment had significant difference with catechol treatment and control,while in the fermentation liquid of phenylalanine did not exist significant differences.3.The effects of catechol on growth,activities of antioxidant enzymes such as catalase?CAT?,superoxide dismutase?SOD?,peroxidase?POD?,glutathione S-transferase?GSH-ST?,glutathione peroxidase?GSH-PX?and glutathione reductase?GR?,as well as expression of melanin biosynthesis genes in R.solani AG-1 IA,were determined by culturing the fungus in catechol-containing nutrient media.The results showed that in addition to the GSH-PX activity was significantly increased in the presence of 50?g/mL catechol,the activities of CAT,POD,SOD,GSH-ST and GR were decreased in the presence of 0 to 50?g/mL catechol,The growth rates of R.solani AG-1 IA on potato dextrose agar?PDA?containing catechol were significantly decreased,whereas the pathogenicity to rice of R.solani AG-1 IA cultured on PDA containing different concentrations of catechol was enhanced when compared to the catechol-free control.The results of quantitative RT-PCR?qRT-PCR?showed that the expression of four melanin biosynthesis genes,i.e.protein tyrosine phosphatase?Ptp?,chorismate mutase?Cm?,peroxidase?Per?and phenol hydroxylase?Ph?,was significantly down-regulated in R.solani AG-1 IA cultured in catechol-containing PDB at 100?g/mL.4.In order to elucidate the functions of phenol 2-monooxygenase gene?RsPhm?and the catalase gene?RsCat?in melanization of R.solani AG-1 IA,the two genes were cloned by routine PCR and RT-PCR techniques.Furthermore,the relative expression levels of these two genes under the stress of catechol were determined by using qRT-PCR technique.Bioinformatic analysis showed that the full-length DNA and cDNA sequences of RsPhm gene were 2 628 bp and 1 983 bp respectively,which encode 660 amino acids.The phylogenetic tree analysis showed that RsPhm gene has a close relationship in different anastomosis groups?AGs?of R.solani,and a certain evolutionary conservation in different fungal species.Results of qRT-PCR indicated that the exogenous catechol could improve the expression of RsPhm gene.Bioinformatic analysis showed that the full-length DNA and cDNA sequences of RsCat gene were 1814 bp and 1395 bp respectively,their open reading frame?ORF?encodes 464 amino acids.The conserved domain analysis showed that the RsCat protein has the domains of catalase_like superfamily and catalase-related immune-response.Phylogenetic tree analysis showed that the RsCat gene in different anastomosis groups?AGs?of R.solani has a high sequence homology.Results of qRT-PCR analysis showed that RsCat gene expression was induced by catechol,the expression levels of RsCat gene were up-regulated with the increase of catechol concentrations,whereas the activity of catalase?CAT?decreased with the increase of catechol concentrations,indicating that the transcriptional expression of RsCat gene and the activity of catalase?CAT?are not corresponding.5.Transcriptome analysis of samples with differences in melanin contents of R.solani AG-1 IA was performed using Illumina Hiseq2000 system.Bioinformatic analysis showed that Reads in Rs?blank=brown?,RsC?50?g/mL of catechol-treatment=dark brown or black?and RsH?300?g/mL hyoscyamine-treatment=white?samples could be accurately matched to the reference genome,indicating that RNA-Seq results and reference genome were reliable.Differential gene expression?DEGs?results showed that 385 genes up-regulated and 208 genes down-regulated in Rs when compared to RsC;69 genes up-regulated and 98 genes down-regulated in Rs when compared to RsH;62 genes up-regulated and 50 genes down-regulated in RsC when compared to RsH;GO enrichment analysis showed that DEGs were involved in 14 molecular functions,16 cellular components and 73 biological processes.KEGG analysis of R.solani AG-1 IA melanin synthesis gene mainly distributed in tyrosine metabolism pathway and melanin synthesis metabolism pathway.The transcriptional levels of the 12 differential genes were detected by qRT-PCR.The results of qRT-PCR analysis showed that the expression patterns of the12 differential genes were consistent with the results of RNA-Seq analysis.Both catechol and hyoscyamine could induce reactive oxygen species?ROS?outbreaks,while ROS was closely related to pathogenicity.RsC and RsH significantly increased when compared to Rs.From the transcriptome,8 melanin genes?AG1IA₀0235,AG1IA₀9755,AG1IA₀1710,AG1IA₀5081,AG1IA₀6379,AG1IA₀9040,AG1IA₀2190,AG1IA₀1614?were screened,and these genes were cloned,sequenced and bioinformatic analyzed,their relative expression levels under catechol stress were detected by qRT-PCR,and their role in the pathway of melanin synthesis metabolism was preliminarily explored.6.The full-length cDNA sequences of melanin synthesis related genes RsCao and RsPhs in R.solani AG-1 IA were cloned by using RT-PCR.The results showed that the full-length cDNA sequences of RsCao and RsPhs were 396 bp and 828 bp,respectively.The expressions of RsCao and RsPhs genes in Pichia pastoris were successfully realized,recombinant P.pastoris transformants could efficiently secrete target proteins of RsCao and RsPhs in BMMY medium containing 1%methanol.By observing the melanin formation of recombinant P.pastoris transformants at different concentrations of catechol in YPD,the results showed that RsCao and RsPhs had different melanin effects,qRT-PCR also showed that the expressions of RsCao and RsPhs genes in R.solani AG-1 IA were different at the different concentrations of catechol.The above results clarify the effect of melanin in the pathogenesis of R.solani AG-1 IA and lay the foundation for the control of rice sheath blight.