Transcriptomic Profiling Reveals the JA and ET Signal Transduction during Field Peanut Pod Rot Pathogen Infection

WANG Mian ,WANG Tong ,CHEN Ming-na ,YANG Zhen ,YU Shan-lin ,CHEN Na ,XU Jing,PAN Li-juan,ZHANG Chao-xin,GUO Xin-gang,CHI Xiao-yuan*

(1. Shandong Peanut Research Institute,Qingdao 266100,China;2. Qingdao Municipal Appearance and Environmental Sanitation Management Center,Qingdao 266071,China;3. Zouping Comprehensive Inspection and Testing Center,Zouping 256200,China)

Abstract:Pod rot,an important disease affecting peanut production,causes serious damage to peanut yield and quality.In this study,RNA sequencing analysis was performed with peanut cultivar Huayu20 kernels that infected with pod rot disease in field,and that uninfected kernels acting as control.As a result,a total of 60,756 unigenes were obtained,among which the expression of 10,147 genes were up-regulated and 4,334 genes were down-regulated.Further,Gene Ontology enrichment analysis showed that the differentially expressed genes (DEGs) were assigned to 24 cellular components,5 molecular functions and 23 biological processes.Through Kyoto Encyclopedia of Genes and Genomes enrichment analysis,a total of 4,291 DEGs were mapped to 34 metabolic pathways.We identified multiple hormone signal transduction pathways that were responsive to pod rot disease infection.Based on the expression pattern of related genes,we speculate that peanuts may resist pod rot disease infection through jasmonic acid-and ethylene-mediated pathways.

Key words:peanut;pod rot disease;transcriptome;JA and ET signal transduction pathway

Pod rot disease causes peanuts to have rotten pods and kernels with dark discoloration,seriously affecting peanut yield and quality[1].In recent years,the prevalence of this disease has intensified in north China.The cause of peanut pod rot disease is complicated and includes pathogenic fungi,parasitic nematodes,soil mites,and environmental factors[2].The composition of pathogenic microorganisms varies in different regions or during different years in the same region,thus the control of peanut pod rot disease is very difficult,and the conventional agronomic measures used to control the disease are limited in their effectiveness[3].Breeding resistant cultivars is the most economic and effective way to control the disease,which requires understanding the mechanisms of disease resistance in peanuts.

The combination and cooperation of multiple mechanisms are involved in the defense of plants against pathogens,which include pathogen recognition,signal transduction,and the activation and expression of resistance genes[4-5].Hormons are known to play essential roles in plant resistance signal transduction pathway[6],for example,JA and ET play an important role in plant resistance[7-9].When plants are not infected by pathogen,JAZ proteins can interact with ET transcription factors to inhibit transcriptional activity in the absence of JA,whereas,when plants are infected by pathogen,JA is present,and EIN3 and EIL1 are released,as soon as starting the transcription[10-11].

RNA-seq can construct a dynamic map of gene expression changes in plants responding to changes in external environmental factors.To date,thousands of research papers have analyzed the transcriptomes of plants under environmental stress.For example,Kim et al.utilized a near isogenic line to study disease resistance in soybean[12].Zhu et al.used RNA sequencing (RNA-Seq) to study the defense transcriptome ofArabidopsisand its response toFusariumoxysporuminfection[13].At present,a number of pathogen stress responsive genes have been identified in wild soybean[12],Arabidopsis[13],maize[14]and strawberry[15],and their roles in stress resistance have been analyzed.

In this study,we employed RNA-Seq technology to analyze peanut kernel samples.The samples included kernels infected with pod rot as well as uninfected kernels,which served as the control samples.We found that a number of genes changed expression levels during pod rot infection.The goal of this study is to understand the mechanisms of peanut disease resistance at the transcription level,discover genes that are responsive to peanut pod rot infection,and determine the roles of the disease resistance genes in the defense process,so that the key genes can be selected for cloning.The results may provide a theoretic basis for the improvement of disease resistance in peanuts through gene engineering.

1 Materials and Methods

1.1 PlantMaterials

The peanut cultivar Huayu20 was used in our experiment.Peanut plants were grown in the Wangcheng street in Laixi Qingdao under natural growth conditions.At the mature stage,peanut kernels that were infected with naturally occurring pod rot were collected.The kernels from three plants were ground into a fine powder and mixed to form one sample;there was a total of two samples prepared for the infected peanut plants.Uninfected peanut kernels were sampled in the same way and served as the control in our experiment.RNA-Seq analysis of the four prepared samples was carried out by Personal Biotechnology (Shanghai,China).

1.2 RNA-Seq analysis

A TruSeq RNA Sample Prep Kit (Illumina,San Diego,CA,USA) was used for mRNA purification and fragmentation.First,mRNA was purified through the binding of oligo-dT that attached to the magnetic beads and poly-A in the mRNAs,and then fragments of approximately 375 nt were obtained after fragmentation.Double strand cDNAs were synthesized after reverse transcription.Blunt end double strand cDNAs were obtained after they were repaired with 3'-5' exonuclease and polymerase.Next,a single A nucleotide was added to the 3' ends of the blunt end DNA fragments.The 3'ends of the adapters that contained tags have a T nucleotide,which provided a complementary overhang for the annealing of the adapters to the DNA fragments.The adapters and DNA fragments were ligated with DNA ligase.The ligated DNAs were then purified using AMPureXP beads.

DNA libraries were selectively enriched and amplified using PCR.The size and distribution of the DNA libraries were tested through quality control.After dilution,the DNA libraries were sequenced.The original sequencing data was saved in the FASTQ format.Software program FastQC was used in data filtering and quality control.De novo RNA-Seq reads assembly was then performed using the Trinity platform.The transcripts obtained through the Trinity assembly were submitted to the protein database NR to perform a BLASTX search.We set thee-value <0.00001 to retrieve top hits.Unigenes were obtained through clustering.The differential expression analysis of unigenes was analyzed using DESeq.The differentially expressed unigenes were identified based on the fold change andP-value.The expression level between two samples was calculated using Fragments Per Kilobase of transcript per Million mapped reads (FPKM) measurements.The fold change andP-value were calculated for each gene.We also used the Benjamini-Hochberg procedure to correct theP-value,which is a widely accepted effective tool.The threshold was set toP-value <0.05 and fold change ≥ 2.The result was not obtained through direct comparison of the FPKM values.The comparison was performed based on the normalized FPKM values between two samples.Blast2GO (http://www.blast2go.com/)was used in Gene Ontology (GO) annotation of the unigenes.An enrichment score with aP-value ≥ 0.05 was set as the threshold for annotation.By comparing the gene expression in our dataset against the genes of the whole genome,enriched GO classification information for the differentially expressed genes (DEGs) in our samples was obtained.The application websites of Blast2GO and KEGG (http://www.genome.jp/kegg/) were used in the analysis.

1.3 Fluorescence quantitative reverse transcription PCR analysis

In order to verify the accuracy of the sequencing result,we randomly selected 21 genes for quantitative reverse transcription PCR (qRT-PCR) analysis.The sequence of the genes was used to design qRTPCR primers (Table 1).To analysis the expression of the DEGs in JA and ET signal transduction pathway,we selected 8 genes for qRT-PCR analysis and the primers were shown in table 1.Total RNA was extracted from both the infected and control kernels,and then reverse transcripted into cDNAs.The cDNA templates were diluted to 0.5～2.0 ng/μL.Preparation of PCR reactions followed the instructions provided by the manufacturer of SYBR Real Time PCR Master Mix(QPK-201) that was used in our test.A final volume of 20 μL was used for each reaction.

Table 1 The primers used in qRT-PCR analysis of DEGs

Table 2 Out puts statistics of peanut transcriptome sequencing

2 Results

2.1 Statistic analysis and functional annotation of RNA-Seq data

In order to discover the change of gene expression level in response to the infection of pod rot in peanut,our samples were sequenced using the Illumina HiSeq 2500 (2×126 bp) platform.A total of 18,525,795,250 nt of data was obtained.The raw data have been submitted to the BioProject:PRJNA202339 and PRJNA396989.After filtering,we obtained 60,756 unigenes that had a total length of 6,223,200 nt,an average length of 1,024 nt,an N50 length of 1,552 nt,and a GC content of 47.74%,shown in table 2.As the length increased,the number of unigenes decreased.

In order to annotate the protein function of unigenes,the sequence of unigenes was submitted to the nucleic acid database NT to perform a BLASTN search.The sequences were also submitted to the protein databases NR,SwissProt,eggNOG,GO,and KEGG Orthology to perform a BLASTX search.As a result,we obtained the annotation information of 60,756,60,756,48,954,8,133,57,974,and 42,584 unigenes from the NT,NR,GO,KO,eggNOG,and Swiss-Port databases,respectively.Annotation information of 60,756 unigenes was obtained.

In order to determine the overall classification of the genes responsive to pod rot infection in peanut,the unigene sequences were used to search the eggNOG database.As a result,21.94% of the unigenes were classified asunknownfunction,17.9% were classified ascommonfunction,8.49% were related to signal transduction,and 7.42% were chaperones and proteins related to post translation modification and protein reversal (Figure 1).It is clear that a substantial number of genes with known or unknown functions responded to pod rot infection in peanut.

Fig.1 EggNOG functional category of all Unigenes in peanut transcriptome

2.2 Screening of DEGs

The DEGs were analyzed using DESeq.The setting was a fold difference ≥ 2 (log2Folder difference=1) and a false discovery rate ≤ 0.001.As a result,a total of 14,482 genes showed differential expression,of which 10,147 genes were up-regulated and 4,334 genes downregulated.For the genes with up-regulated expression,their increased expression level varied from 1-to 14-fold.For genes with down-regulated expression,most decreased 1-to 5-fold,but a few genes decreased over 8-fold (Table 3).Specifically,genes c249_g1_i1 and c292_g1_i1 decreased over 14-fold.The above findings indicate that a large number of genes exhibited differential expression in response to peanut pod rot infection,and many of them were new genes related to disease resistance,suggesting the response of peanut to pod rot infection is a complicated process that involves a number of genes.

Table 3 List of up-regulated or down-regulated 30 genes in peanut response to pod rot

2.3 Fluorescence quantitative RT-PCR

In order to verify the reliability of the RNA-Seq data,we randomly selected 21 DEGs for qRT-PCR;the results are shown in Figure 2.Of the analyzed genes,6 were up-regulated,13 were down-regulated,and 2 did not show any obvious change.The fold change of up-or down-regulated genes was basically consistent with that of the sequencing result obtained through RNA-Seq analysis.Thus,the RNA-Seq data can be used for further analysis.

2.4 GO analysis of DEGs

The DEGs were submitted to the GO database for functional annotation to determine the biological function of the genes.That the DEGs were classified into 24 cellular components,5 molecular functions,and 23 biological processes (Figure 3).Through annotation analysis,we found that peanut pod rot disease clearly affected three types of proteins in different physiological processes.

Fig.2 Verification of the RNA-seq results by qRT-PCR

2.5 KEGG pathway analysis of DEGs

In order to understand how the DEGs are involved in metabolic pathways,we submitted the genes to the KO databasepathways with aP-value ≤ 0.05.The searching result showed that 4,291 DEGs were assigned to 34 metabolic pathways,among which 442 genes were involved in carbohydrate metabolism,383 genes were involved in signal transduction pathways,321 genes were involved in cellular metabolism,and 316 genes were involved in amino acid metabolism(Figure 4).Many of the DEGs were found to be involved in genetic transformation.

Fig.3 GO functional annotation and classification of DEGs under peanut pod rot pathogen

Fig.4 KEGG pathway enrichment analysis of DEGs under peanut pod rot pathogen

The hormone signal transduction pathways in response to peanut pod rot disease infection were further screened.We found that 70 DEGs were involved in multiple plant hormone signal transduction pathways,of which 43 genes were down-regulated (Figure 5).For instance,in the salicylic acid (SA)-mediated disease resistance pathway,the expression of twoNPR1 and fiveTGAgenes decreased,while no noticeable change in expression level was observed in thePRgene.In the same way,we analyzed the differential expression of genes in other pathways related to disease resistance.For example,a total of 17 DEGs were found in the mitogenactivated protein kinase (MAPK) signal transduction pathway,of which 13 genes were up-regulated and 4 genes were down-regulated.In the plant-pathogen interaction pathway,we found 50 DEGs,of which 33 genes were up-regulated and 17 genes were down-regulated.Overall,in those two pathways,the down-regulated genes included 11 genes in the MAPK family,6 genes coding for serine/threonine kinase and their sub unit,5 of 6 heat shock protein genes,4 WRKY genes,and genes coding for other kinase and receptor proteins.The results indicated that after peanut pod rot infection occurs,the genes in the disease resistance pathways were down-regulated,suggesting that the reduced ability of the plant to express resistance genes results in disorders in the disease resistance pathways,and as a result the entire plant is damaged.This finding may account for the lack of effective pod rot resistance in peanut,and may also be one of the reasons for the high fatality rate of this disease.

Fig.5 Diagram of hormone signal transduction network in peanut pod rot

2.6 qRT-PCR of the DEGs in JA and ET signal transduction pathway

In order to verify the expression of the DEGs in JA and ET signal transduction pathway,we selected 8 DEGs for qRT-PCR,the results are shown in Figure 6.In the jasmonic acid (JA)-mediated disease resistance pathway,the expression of twoJAR1 genes and theJAZgene were down-regulated.In the ethylene-mediated disease resistance pathway,the expression of oneETRgene decreased,whereas the expression of twoEIN3 genes and oneEBF1-2 gene increased.It is clear that the expression level of the majority of the genes in disease resistance pathways change during pod rot infection,suggesting that pod rot infection may be a consequence of the low expression level of those genes.

Fig.6 Verification of the RNA-seq results by qRT-PCR

3 Discussion

The plant genome project provided a large amount of data and supplied a basis for the in-depth study of plant genomes.To date,the entire genome ofArabidopsis[16],wheat[17-18],rice[19],maize[14],season[20],and soybean[21]has been sequenced.The correlation of genes in these networks has also been analyzed[22],and the data has become increasingly important for understanding the molecular mechanisms that control important traits.

In order to obtain complete and accurate data about the type and number of genes involved in pod rot resistance in peanut kernels,infected and uninfected kernels from plants of the cultivar Huayu20 grown in the field under natural growth conditions were subjected to RNASeq analysis.The disease resistance-related pathways and genes responsive to pod rot infection were screened.The role and expression of the genes in disease resistance-related pathways,including the plant hormone signal transduction pathway,plant-pathogen interaction pathway,and MAPK signal transduction pathway,were analyzed to find the key resistance genes and resistance pathways.The results of our study may benefit the breeding of pod rot resistant cultivars in peanut.

3.1 The RNA-Seq data of peanut pod rot responsive transcriptome is reliable

The genome of diploid wild peanut and cultivated peanut have been sequenced.But part gene annotations on the whole genome sequence are still lack.Therefore,utilizing RNA-Seq analysis to study gene structure and function is still an important method to discover new genes and determine the role of genes and their regulation in peanut[23-24].We performed RNA-Seq analysis of pod rot infected peanut kernels as well as uninfected kernels,and verified the change at the expression level of the DEGs by qRT-PCR analysis of randomly selected genes.We analyzed the role of the genes and their expression pattern under pod rot stress and found that the DEGs mainly play a role in protein kinase activity,defense response to pathogens,and response to SA and JA stimuli.The results indicated that,at the transcription level,peanut enhances disease resistance mainly through regulating the protein kinase activity,plant hormones and resistance signal transduction,and increasing the expression of genes for synthesizing substances related to disease resistance.This result is in agreement with results from studies on disease resistance in other plants previously reported by other authors.We annotated unigenes in the eggNOG database and found that 21.94% of the genes were categorized as unknown function,which means a large number of genes with unknown functions are expressed during pod rot infection.Thus,it is possible to find new resistance genes within that group of genes.Our results are in agreement with Tan's finding that many genes with unknown functions were expressed during the defense response ofBrassicanapusagainstsclerotiniastem rot[25].Those results indicated that plant disease resistance is a very complicated process.

3.2 Peanut resistance to pod rot disease may be mediated by JA and ET signal transduction pathway

The defense response to pathogen infection tends to change the endogenous hormone level in plants.Through KEGG analysis,we found that 70 DEGs were involved in hormone signal transduction during pod rot infection.The signal transduction pathways may promote or suppress each other.For example,they may interact and work together to achieve resistance to disease.SA is a signaling molecule that rapidly accumulates after pathogen infection,and gives rise to a hypersensitive response that induces a systemic and broad spectrum resistance in plants to defend themselves against pathogens[11,26].In our study,in the SA-mediated disease resistance pathway,two SA receptorNPR1 genes and fiveTGAgenes were down-regulated,while thePRgene did not show a clear change in the expression level between the infected and uninfected kernel samples.Based on this finding,we conclude that the SA-mediated resistance pathway did not start up during the infection of pod rot in peanut.Alternatively,the regulation mechanism in this pathway was suppressed,and thus the peanut plants were highly susceptible to pod rot disease.

The JA signal transduction pathway mediated plant disease resistance pathway has two branches,one branch is correlated with the ET-mediated disease resistance pathway through the ethylene response factor pathway to resist pathogen infection,and the other branch is inversely correlated with the SA pathway[27-29].In our study,the expression of twoJAR1 genes was up-regulated before pod rot infection and the expression of theJAZgene was down-regulated after infection in the JA-mediated disease resistance pathway.In the ET-mediated disease resistance pathway,the expression of the ethylene receptorETRgene,twoEIN3 genes,and oneEBF1-2 gene were up-regulated after pod rot infection.These results suggest that during pod rot infection,the accumulated JA mediated the degradation of the transcription suppressor jasmonate ZIM domain protein,which led to the expression of the downstream transcription factor genesEIN3 and anEBF1-2.Thus,the responsive reaction of peanut to pod rot infection took effect through the JA and ETmediated disease resistance pathways.

3.3 Multiple disease-resistance gene families are involved in the defense of peanut against pod rot infection

A number of disease resistance genes in plants have been cloned,and many of these genes,such as the genes coded for the nucleotide binding site leucine rich repeat (NBS-LRR) protein[30],pathogenesis-related (PR) protein[31],MAPK[32],and WRKY[33],have conserved regions.NBS-LRR,receptor like kinase,and PR proteins contribute to disease resistance through recognizing pathogen proteins[34].MAKK,G-proteins,calcium-dependent protein kinase,and mildew locus O (MLO) respond to biotic and abiotic stresses through signal transduction.WRKY and MYB are important transcription factors that respond to environmental stress[35].In addition,ATP binding cassette (ABC) and amino acid permeases (AAP) transporters,and plant hormone and secondary metabolites also play important roles in plant disease resistance[36].

Through the functional annotation of DEGs obtained by RNA-Seq analysis,we found that during pod rot infection,the DEGs included 11 genes in the MAPK family and genes coding for the WRKY transcription factor,disease resistance-related proteins NBS-LRR,PR,UDT glucuronosyltransferase and cytochrome P450,signal transduction-related proteins such as G-protein and MLO protein,ABC and AAP transporters,and proteins involved in the synthesis of JA,ET,lignin,and isoprene.These findings suggest that the DEGs coding for or regulating those proteins and transcription factors may be related to pod rot disease resistance in peanut.

Acknowledgments

This study was supported by grants from the National Natural Science Foundation of China (31701464),China Agriculture Research System (CARS-13),Taishan Scholar Project Funding (tsnq201812121),the Natural Science Fund of Shangdong Province (ZR2020MC103),Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2016B02,CXGC2018E21),Major Scientific and Technological Innovation Projects in Shandong Province,the Key-Area Research and Development Program of Guangdong Province (2020B020219003);Qingdao People's Livelihood Science and Techonology Program(20-3-4-26-nsh);the Key Research and Development Program of Linyi (2019YD009).

Accessibility

The sequences data from the four samples obtained are available in the NCBI Sequence Read Archive as bioproject:PRJNA202339 and PRJNA396989.