Mining And Functional Studies Of Peptides And Proteins With Disease Resistance In Maize

Plant diseases can threaten food security by reducing crop yield.Therefore,the selection of disease-resistant varieties is one of the major goals in crop breeding.Discovery of novel small peptides and proteins related to disease immunity is of great theoretical and practical significance for crop improvement.Peptide is a kind of small molecule substance composed of 2~100 amino acids,which plays important roles in various life activities of organisms and has high development and application value.Previous studies mostly focused on conventional peptides(CPs)derived from conventional coding regions.Latest researches showed that non-conventional peptides(NCPs),which are derived from the regions previously deemed as noncoding,also play key roles in a variety of biological activities,including development and immune response.However,the lack of extraction and identification methods of large-scale NCPs greatly hampers the research of plant NCPs,and also seriously limit our understanding of plant NCPs and their application.Centering on the demand for large-scale discovery of plant NCPs,we developed a peptidogenomic pipeline for the first time in this study,and applied the pipeline to both monocots(maize)and dicot(Arabidopsis)to identify their corresponding NCPs in a large-scale.Based on the developed peptidogenomic pipeline,a comparative peptidogenomic system was established to identify the NCPs induced by pathogen-associated molecular pattern(PAMP)in maize.Subsequent functional studies initially confirmed the positive roles of NCPs in regulating the innate immune response and disease resistance in maize.In addition,southern corn rust resistance proteins were identified by comparative proteomics analysis.Functional analysis of one of the candidate proteins(Zm REM1.3)indicated its positive role in P.polysora resistance,and investigated the potential resistance mechanisms.The main results are as follows:1.This study developed a peptidogenomic pipeline for the first time.Heat stabilization in water bath and plant protease inhibitor were introduced to avoid large protein and endogenous peptides degradation during the extraction.In addition,10 KDa centrifuge filter was used for peptide enrichment.The enriched endogenous peptides were analyzed using high-throughput mass spectrometry,of which the results were searched Ensembl protein database and genome six-frame translation database,respectively.Highly reliable endogenous peptides were collected and matched to the corresponding genomic locations,and those matched with multiple genomic locations were discarded.Finally,endogenous peptides from conventional coding regions were defined as CPs according to genome annotation,and endogenous peptides from the regions previously regarded as noncoding(intergenic regions,UTRs,introns,and junctions)were defined as NCPs.2.1,993 NCPs and 844 CPs were identified in maize using the developed peptidogenomic pipeline.Peptide length of NCPs was significantly shorter than that of CPs,and the molecular weight of NCPs was also smaller than that of CPs.These results showed that NCPs and CPs have different molecular characteristics.By analyzing the genomic distribution of NCPs and CPs,it was found that NCPs presented distinct distribution patterns from CPs.Most CPs were distributed near telomeres on chromosomes,while NCPs were homogeneously located between the centromeres and telomeres.Further analysis of the sources of NCPs,we found that the sources of NCPs were derived from the intergenic region,introns,UTRs,and junction regions of different gene elements.These results revealed that a much larger portion of the plant genome thought to be untranslated is translated with direct evidence at protein translation level.3.Three different experimental methods were used to validate the identified NCPs.First,the synthetic peptide mass spectrometry and the experimental mass spectrometry were verified by the synthetic method,and the results showed that the experimental mass spectrometry and the corresponding synthetic peptide mass spectrometry were highly consistent.Next,transcriptomics analyses of m RNA,circ RNA,lnc RNA and small RNA were performed,and the results showed that 1806(90.62%)NCPs were supported by transcriptional data.Finally,ribosome profiling(ribo-seq)analysis was used to validate the identified NCPs.The results showed that 732 NCPs could be validated by the results of ribo-seq.These results demonstrated the reliability of our developed peptidgenomic pipeline.In order to further study the applicability of the developed peptidogenomics,the pipeline was further successfully applied to the dicot model plant Arabidopsis.As results,1860 NCPs and 2363 CPs were identified in Arabidopsis.Analysis showed that the peptide length of NCPs identified in Arabidopsis was also significantly shorter than that of CPs,and NCPs identified in Arabidopsis were also originated from intergenic regions,intron regions,UTRs,and even junction regions.The results showed that translations in the regions previously annotated as untranslated are common in both monocot and dicot plants,although they may have different translation patterns.The peptidogenomics pipeline can also be applied to both monocot(maize)and dicot(Arabidopsis)plants,proving the wide applicability of the developed peptidogenomic pipeline.4.Further analysis combined with GWAS showed that NCPs were significantly enriched within those regions associated with phenotype variation(kernel trait and disease resistance)and undergone domestication,suggesting the important role of NCPs in maize evolution and complex traits,especially in maize disease resistance.To further study the interaction between maize NCPs and disease resistance,a comparative peptidogenomic system was established.Plant innate immune elicitor flg22(PAMP)was used to treat maize,and then the changes of maize endogenous peptides induced by PAMP were investigated.A total of 662 differentially accumulated endogenous peptides were identified,including 451 CPs and 211 NCPs.At 30 minutes and 24 hours after treatment,428 differentially accumulated endogenous peptides were identified,respectively.There were 194 differentially accumulated endogenous peptides identified at both 30 minutes and 24 hours after treatment.Among these differentially accumulated peptides,6 NCPs were up-regulated and 7 NCPs were induced at 30 minutes and 24 hours after treatment.The identification of PAMP-induced NCPs in maize has laid a foundation for the research on the function of NCPs in plant innate immune response.5.In order to investigate the role of NCPs induced by PAMP in plant innate immune response and disease resistance,immune-related parameters of 13 up accumulated and induced NCPs induced by PAMP were determined.The results showed that 2 NCPs played important roles in plant innate immune response: the one which presented multiple disease resistance in maize was named as NCP-1.The results showed that NCP-1 could promote the accumulation of reactive oxygen species(ROS),the deposition of callose and the expression of defense-related genes.Finally,through the identification of the resistance of southern corn leaf blight,root rot and Curvularia lunata,NCP-1 can significantly improve the resistance of multiple diseases in maize.The above results indicated that NCP-1 can be used as a plant immune signal molecule to regulate the innate immune response of plants,thereby inducing disease resistance.This study is the first report of NCPs in the innate immune response,providing a new idea for the functional research and disease resistance research of plants.6.To identified southern corn rust resistant protein,comparative proteomic analysis was performed using leaves of susceptible(Lx9801)and resistant(P178)inbred lines after inoculation with P.polysora and mock inoculation.A total of 6,612 proteins were identified by isotope labeled relative and absolute quantification(i TRAQ)proteomics,among which,1,489 proteins were differentially accumulated in resistant inbred lines,and 1,035 proteins were differentially accumulated in susceptible inbred lines.GO enrichment analysis showed that these differentially accumulated proteins were enriched in various biological processes.The resistant group was mainly enriched in metabolic process,cellular process,biological regulation and response to stimulus,while the susceptible group was mainly enriched in metabolic process,cellular process,and response to stimulus.KEGG enrichment analysis showed that these differentially accumulated proteins were mainly enriched in the carbon fixation pathway,ribosomal metabolism and photosynthesis in the resistance group,while those in the susceptible group were mainly enriched in the carbon fixation pathway,metabolism pathway and carbon fixation pathway.7.Among these differentially accumulated proteins,3 defense related candidate proteins were selected for further study,the results confirmed the important role of one candidate protein in southern corn rust.q RT-PCR analysis showed that the expression of ZMREM1.3 was induced not only by P.polysora,but also by the plant hormones salicylic acid(SA)and jasmonate acid(JA).Furthermore,Zm REM1.3 was overexpressed in maize using transgenic technology.q RT-PCR and western blot analysis showed that the expression level of Zm REM1.3 in transgenic positive lines was significantly higher than that in corresponding negative lines.After inoculation of P.polysora in transgenic lines,it was found that the resistance of transgenic maize plants overexpressing Zm REM1.3 to P.polysora was enhanced.In addition,the positive role of Zm REM1.3 in P.polysora resistance was confirmed by the Mu transposon mutant analysis.Further analysis showed that the SA and JA contents of Zm REM1.3 overexpressed plants were significantly increased,and the expression level of defense-related genes in Zm REM1.3 overexpressed maize lines was significantly higher than that of corresponding non-transgenic control lines after the infection of SCR.In summary,our results suggested that Zm REM1.3 may through SA/JA-mediated defense signaling pathways induce the expression level of defense related genes,positively regulate P.polysora resistance.In summary,this study developed a peptidogenomic pipeline,providing a new method for the discovery of plant NCPs,and opening up a new avenue for plant NCPs research.Large-scale NCPs identification of monocot and dicot has shown that a large portion of the plant genome which was previously regarded as untranslated is translatable.This is of great significance in the study of functional genomics,and will provide a new focus for the study of plant functional genomics.Through the research on the function of NCPs in the innate immune response of plants,the important role of NCPs in the innate immune response of plants was confirmed for the first time,which provided a new idea and direction for plant disease resistance research and disease resistance breeding.Southern corn rust resistant proteins(Zm REM1.3)were identified by proteomic techniques,and the positive role and potential mechanism in southern corn rust resistance was experimentally confirmed.It provides a new resistant resource for maize disease resistance breeding.