| Rice blast is caused by the fungal pathogen Magnaporthe oryzae and is the most destructive disease of rice and allures the deepest understanding.With the accessibility of both genome sequences and the mighty molecular genetic tools,the rice-M.oryzae pathosystem has become a model system for studying plant-pathogen interactions.However,in the key early stages of rice-M.oryzae interaction,especially the rice defense mechanism and molecular processes,are not yet clear.Quantitative proteomics based on iTRAQ has become a powerful tool for investigating the complex molecular processes in the era of the rice-M.oryzae interaction with quickly developing of research techniques in proteomics.However,no enough data are available for the rice proteome in response to M.oryzae.To date,some confer broad-spectrum resistance genes,such as Pi2,Pi9,Piz-t and Pigm,have been mapped on Pi2/9 loci.These genes,therefore,have been extensively used in MAS breeding program to improve blast resistance in rice.Nevertheless,owing to some genetic distances between SSR markers and candidate gene,or some markers cannot truly reflect the gene ontology,these markers prone to mismatch or missed selection in resistance breeding,and are not suitable for large-scale molecular breeding.Additionally,the genetic information of the varieties and breeding materials used in the Pi2/9 locus is also difficult to determine due to the absence of available markers.Thus,it will be of great importance in developing the allelic-specific markers on Pi2/9 loci for breeding.In order to address the above issues,the following three aspects of research had been carried out used a combination of proteomics based on iTRAQ,bioinformatics,targeted metabolomics and molecular breeding.Firstly,to explore the molecular mechanisms of incompatible interaction(resistance)and compatible interaction(susceptibility)of Piz-t,we compared the common and specific DEPs in two interactions.Secondly,by comparing with two interactions systems(KJ201-Piz-t/KJ201-NPB and KJ201-Piz-t/RB22-Piz-t)of DEPs,we investigated the conservative elements of NPB-Piz-t incompatible and compatible interaction,and with the help of targeted metabolomics,RT-PCR,and Western-Blot verification method to study the expression pattern of three proteins under incompatible and compatible interaction.Thirdly,the study developed Pi2,Piz-t,and Pi9 allelic-specific markers bybioinformatics methods,and utilized the genes or markers of Piz-t locus to classify blast resistance genotype of 434 rice varieties and apply in breeding practice.The main results are as follows:1.118 and 119 DEPs were detected in KJ201-Piz-t/CK-Piz-t and RB22-Piz-t/CK-Piz-t,respectively.Both of them have 59 similar expression characteristics of common DEPs,and the main functions involved are carbon metabolisms,biosynthesis of amino acids,photosynthesis,carbon fixation in photosynthesis organism and RNA degradation,which may belong to the recognition related proteins associated with the molecular pattern of pathogens.In KJ201-Piz-t/CK-Piz-t,there are 58 specific DEPs that mainly involve TCA,photosynthesis-antenna proteins,amino acid metabolism,and other secondary metabolism such as phenyl propane synthesis.RB22-Piz-t/CK-Piz-t has 60 specific DEPs with most of them were up-regulated,which mostly include carbon metabolism,dicarboxylic acid metabolism,pyruvate metabolism,TCA,oxidative phosphorylation,linolenic acid metabolism and some secondary metabolites synthesis.The PPI shows that the main function of common DEPsis to suppress of photosystem in 24hpi,to accelerate lipid,carbohydrate and energy metabolismand decrease the co-protein translation modificationin 48hpi and 72hpi.The proteins in KJ201-Piz-t/CK-Piz-t regulate the photosynthetic system and life activities in 24 hpi and 48 hpi,and gradually restored normal function to meet the demand of synthesis of secondary metabolites in 72 hpi.While in RB22-Piz-t/CK-Piz-t,from 24 hpi to 72 hpi,oxidative decomposition metabolism is the main activity.Therefore,the repair function of photosystem may be the most significantly regulatory pathway of incompatible interaction of KJ201-Piz-t.2.There were 93 DEPs to be identified in KJ201-Piz-t/KJ201-NPB,involved in sulfur metabolism,carbon fixation,porphyrin and chlorophyll synthesis,flavone and flavonol synthesis,arginine synthesis.In the contrast of KJ201-Piz-t/RB22-Piz-t,54 DEPs involved in dicarboxyl ic acid metabolism,the pentose phosphate pathway,alpha-linolenic acid metabolism,fatty acid metabolism and amino acyl-tRNA biosynthesis.GO and PPI showed that the difference of DEPs in the contrast of KJ201-Piz-t to two kinds of compatible interaction were obvious,and more proteins and metabolic pathways were affected in KJ201-Piz-t/KJ201-NPB in 24 hpi and 48 hpi,which indicated the difference in response intensity between different compatible interaction systems.There were also 10 common DEPs in two compatible interactions participated in resistance reaction and energy metabolism process,and 8 of them had the same expression pattern,which belonged to the regulatory proteins involved in the NPB-Piz-t incompatibility interaction.3.Three representative proteins gi|59800021,gi|20196,and gi|222637272 were chose to analyze their associated proteins,interactions and expression at different levels of RNA and protein(metabolites)by using rRT-PCR,targeted metabolomics,and Western blot.The results revealed that RNA and protein expression levels of gi|59800021 and gi|20196 in incompatible interaction were higher than compatible interaction in 24 hpi and 48 hpi.There was a significant change in the content of cysteine(CYS),S-adenosyl-homocysteine(SAH)and methyl-thioadenosine(MTA)related to gi|222637272 at various time-points in incompatible interaction by comparison to compatible interaction.4.Based upon the NBS2-5'UTR sequence of Pi9 promoter,referring to the sequences of other alleles including the sequence characteristics of Pi2,Piz-t,NPB and other 26 representative varieties,we developed the 9-pro marker which has 27 bp difference between Pi9 and Pi2/z-t and 10 bp difference between Pi9 and other allele.Therefore,this marker distinguished Pi9 and Pi2/z-t well but not Pi2 and Piz-t.There were eight amino-acid differences between Pi2 and Piz-t and within the first 6 of them,there were two sites could be recognized by restriction enzyme Pstl and Hinfl by comparing Pi2,Pi9,Piz-t,and Pi2 of Nipponbare and other alleles.Upon this,we developed amarker named CAPS(cleaved amplified polymorphic sequence)to identify Pi2 gene,labeled Pi2-LRR.2-LRR-F/2-LRR-R in Pi2 and Piz-t separately amplified 436bp and 439bp segments,and further by using PstI or Hinfl restriction enzyme we cut PCR products and obtained fragments with significant differences.The developed markers and materials identified containing Pi2 and Piz-t aforesaid were confirmed by indoor inoculation test.The distribution of the resistance genes labelled by 9-Pro and 2-LRR markers in the F2 population matched with the distribution in materials inoculatedwith strain.The resistance level of materials containing Pi2 and Piz-t was equal to the corresponding positive parental control C101A51 and Torid-1 at least.5?We transferred Pi9 and Pi2 into eight excellent restorer lines to obtain resistance improved lines.The study results indicated that the resistance level of the developed lines was improved dramatically enhanced by inoculation with 19 representative rice blast dominant strains collected from Fujian province or the field-resistance identification in Chadi town,Shang hang county,and no significant difference was found of the agronomic traits between the five confirmed lines and their original parents.We used 9-Pro and 2-LRR markers developed to identify the 434 China's major rice varieties or breeding materials.The results demonstrated that these materials did not contain Pi9,but one contained Pi2 and fourteen contained Piz-t;therefore,the three resistance genes had not been widely used in production and were likely to be worthy of utilization in resistance breeding in the future. |
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