Molecular Response To The Interaction Between Sugarcane And Ustilago Scitaminea

Sugarcane,an important sugar crop in many tropical and sub-tropical areas, accounts almost 90%sugar production in China.As the main sugar crop and energy crop with its high bio-mass,sugarcane has been the hot spot in recent science research. Sugarcane smut induced by Ustilago scitaminea Syd.is one of the most important fungal diseases in Mainland China and worldwide,which causes serious yield loss and results in no millable stalk and elimination of some leading varieties for the reason of being sensitive to smut.Sugarcane breeding of varieties resistant to smut is the most effective measure for disease management and molecular breeding is potentially an important approach.Accordingly,the investigation of molecuiar response to interaction between sugarcane and U.scitaminea is imperative.In this study,molecular marker techniques of RAPD,SRAP and ITS are adopted for analysis of the differentiation and genetic diversity of 23 isolates.These isolates were collected from sugarcane infected by U.scitaminea in different sugarcane clones cultivated in six different provinces including Fujian,Yunnan,Jiangxi,Guangdong, Hainan and Guangxi.Then taking NCo376 and Ya71-374 as plant materials,we mainly focus on the molecular response to sugarcane/U.scitaminea interaction via modern molecular biology techniques,such as cDNA microarray,2-DE,MALDI-TOF-TOF /MS and Real-time qPCR.Also RAPD and BSA were occupied to identify the molecular marker related to smut resistance.The results indicated that the molecular differentiation of U.scitaminea was associated with its geographical origins,but not to its host origins.In other words, there was not a co-evolution mechanism between sugarcane and the smut pathogen.It also showed that ITS marker is not suitable for the phylogenetic relationship analysis of different sugarcane smut isolates.Then,in pin-prick inoculation,race 1 of U.scitaminea was selected for preparation of the teliospore suspension at the concentration of 5×10⁶ spores/mL and taken as inoculation source.The differentially expressed genes and differentially expressed proteins from sugarcane/U.scitaminea interaction were investigated.The outcome from cDNA microarray hybridization was further validated by Real-time qPCR test.Based on cDNA microarray hybridization, we have totally obtained 36 genes which up-regulated in sugarcane/U.scitaminea interaction.They involved in several metabolism pathways,such as photosynthesis, ion transport and nucleotide metabolism.Some genes correlated with transcription factors,proteins synthesis and modulation,and cellular signal transduction were also up-regulated.Via MALDI-TOF-TOF/MS and bioinfprmatics,20 differentially expressed proteins before and after the inoculation were found to relate to protein synthesis,signal transduction,photosynthesis and so on.Among them,there were five differentially expressed proteins which hypothesized to have function in sugarcane smut resistance,including microtubule associated proteins,cytochrome-c peroxidase, 2-oxo-acid dehydrogenase,oxidoreductases and protective antigen.Still,three proteins were function unknown,all of which have no significantly homologous to any proteins deposited in proteins database.Interestingly,one NBS-type resistance gene and one NBS-type resistance protein were both found to be up-regulated in the process of sugarcane/U.scitaminea interaction.It is most probable that they play a great important role in sugarcane smut resistance.According to the conserved motifs in the NBS regions of the three typical NBS-LRR type cloned gene RPS2,N and L6,five degenerate and one non-degenerate primers were designed to correspond to the P-loop motif in the sense direction,while nine degenerate plus one non-degenerate primers were made corresponding to the HD motif in the anti-sense direction.Then,the homologous PCR was used to amplify NBS sequence from genomic DNA and cDNA of NCo376.In all,eleven RGA were cloned, five from DNA and six from cDNA,all of which the deduced amino acids encoded all the four internal motifs characteristic of the NBS regions,including P-loop, Kinase-2a,Kinase-3a and HD.The corresponding Accession numbers in NCBI database were EF059973,EF059974,EF059975,EF059976,EF059977,EF155648, EF155649,EF155650,EF155651,EF155652 and EF155653.Clustering analysis showed that all RGA cloned in this study showed specificity to the non-TIR-NBS-LRR group based on the facts that all RGA cloned in this study and the other two typical non-TIR-NBS-LRR type gene RPS2 and XA1 branched to one group,while the two typical TIR-NBS-LRR genes N and L6 branched to the other group.Further,amino acid sequences alignment revealed that all of them contained the residue W in LLVLDDV(W/D)motif.It is suggested that only non-TIR-NBS-LRR but not TIR-NBS-LRR type resistance genes existed in sugarcane genome to some extent. Then one RGA termed PIC(Accession number:EF059974)was selected randomly for function validation.The result showed that the expression of PIC gene can to some extent be influenced by U.scitaminea,SA and H₂O₂.Together with the characters of constitutive expression and tissue-specific,the RGA cloned in this study was most probably resistance related.While with the characteristics of NBS,one of them termed cRGA6(Accession number:EF155648)had no significant similarity to any of the cloned genes except one NBS-LRR type resistance gene from Zea mays.It suggested that it possibly the partial sequence of a new NBS-LRR gene in sugarcane.Therefore,the full-length cDNA of EF155648,which termed as SNLR gene,was cloned through RACE method and its expression profile under the treatment of U.scitaminea,SA and H₂O₂ were investigated by Real-time qPCR.The expression of SNLR gene was influenced by the fungus,SA and H₂O₂.Under the treatment of the fungus,H₂O₂ and SA,the expression patterns of SNLR gene were "down-up","down in the whole process" and "up-down", respectively.Maybe the expression of SNLR gene occurs both via an H₂O₂-and SA-dependent pathway.At the same time,SNLR gene was found to be highly expressed in leaves,mildly in stalks and slightly in roots,which indicated its relation to smut resistance in this aspect.The effect of SNLR gene in the improvement of sugarcane smut resistance should be further studied by over expression in sugarcane or by gene knockout etc.Also,RAPD and BSA were occupied together for screening of molecular marker linked to smut resistance.A polymorphic fragment of smut resistant/susceptible genotype amplified by RAPD was screened and converted into a dominant SCAR marker SC619 successfully.It would be potentially useful for identification of sugarcane smut resistance.From all the above,we know that the molecular response to the interaction between sugarcane and U.scitaminea occurred simultaneously both at the transcription and translation level.Sugarcane smut resistance forms in the process of molecular adaption, due to the co-function of numerous genes and diverse proteins.So this research can provide the foundation theories for uncovering the molecular mechanism of sugarcane smut resistance.Besides,the SNLR gene and SC619 could potentially be used in genetic modification and marker-assisted selection,respectively.The research on genetic diversity of U.scitaminea and its relation to geographical origin and host origin provide the basis for the selection of pathogen in resistance identification and the arrangement of variety distribution.