Transcriptional Characterization of Cysteine Protease Genes against Xanthomonas oryzae pv. oryzae in Rice (Oryza sativa L.)

다국어 초록 (Multilingual Abstract)

This study aimed to examine cysteine protease genes (CPs) at molecular and genetic level in rice. Gain-of-function and down-regulation analysis were performed to uncover their biological significance especially in response to biotic stress. The whole-transcriptome shotgun sequencing screen of the OsCP3 overexpression and RNAi-mediated knockdown transgenic lines revealed the intrinsic transcriptional dynamics during early interaction between Xanthomonas oryzae pv. oryzae and rice.

In chapter 1, database-searching for rice cysteine protease resulted in the identification of six families of OsCPs. Each family is distinctly represented by different proteolytic enzyme, including the 51 papain-like gene members of C1 family, single calpain-2-type member of C2, 7 ubiquitinyl hydrolase-L1 members of C12, 6 legumain members of C13, 2 caspase-1 members of C14, and 7 pyroglutamyl-peptidase 1 members of C15. Subsequent in-depth molecular analysis of cysteine protease members revealed the genomic rearrangement and expansion especially of papain-like cysteine proteases (C1 family) as indicated by a large number of members distributed across 12 chromosomes of the rice genome, as well as extensive variation in intron number and phase. Further investigation of physicochemical properties, gene and protein structure, motif, and cis-element highlighted the common and distinct characteristics of members of each family. It was found that 59.2% of the total numbers of cysteine protease in rice are stable. Protein type prediction based on subcellular localization revealed that all members of C1A, C13, C14, and C15 are globular proteins, whereas single member of C2 and 2 members of C12 are membrane proteins. Highest number and most variable motifs are evident in C1 and C2 family members, while the rest of CP families shared distinct set of few motifs. Gene expression analysis of selected C1 members including OsCP2 (LOC_Os01g67980), OsCP3 (LOC_Os05g01810), and OsCP5 (LOC_Os02g27030) showed that both OsCP2 and OsCP3 are tissue-specific, while OsCP5 is constitutively expressed in all tissues. Transcripts of all three genes were highly induced by Xanthomonas oryzae pv. oryzae (Xoo) race K3a, 100 μM salicylic acid (SA), and 100 μM methyl jasmonate (MeJA) at 12 hours post-inoculation. Moreover, mRNA of OsCP2 and OsCP3 were activated 6 to 12 hours after imposing salinity stress, while OsCP5 was up-regulated by both heat and salt at 6 hours after treatment.

In Chapter 2, three selected papain-like cysteine protease members were cloned for functional analysis in rice. Evolutionary divergence analysis revealed that OsCP2 (LOC_01g73980.1) and OsCP3 (LOC_Os05g01810) are related (0.229) and are both associated (0.401 and 0.400) to XCP2 (At1g20850), a papain-type cysteine endopeptidase also known as xylem cysteine peptidase 2 in Arabidopsis, while OsCP5 (LOC_Os02g27030) is related (0.404) to RD19B (At2g21430). Biological investigation of cysteine protease was carried out using overexpression and RNAi transgenic lines. The total number of overexpression transgenic rice generated was six in OsCP2ox, nine in OsCP3ox, and nine in OsCP5ox. Moreover, five OsCP3RNAi transgenic rice plants were also developed. Stable inheritance of each transgene to T1 generation was verified by resistance to hygromycin for overexpression transgenic lines and phosphinothricin for RNAi lines with segregation that conformed (p<0.05) to 3:1 Mendelian ratio. Disease screening with Xoo race K3a resulted in significantly (p<0.05) shorter lesion length (OsCP2ox, 6.82 – 9.13 cm; OsCP3ox, 5.55 – 7.49 cm; and OsCP5ox, 5.40 – 5.68 cm) than that in Dongjin (16.07cm), while OsCP3RNAi lines exhibited significantly longer lesions (17.1 – 18.3 cm) than overexpression lines. Bacterial growth analysis taken at 0, 7, and 14 days post-inoculation (dpi) strongly corroborated with the phenotype data. This indicates that OsCP genes confered resistance to Xoo. Although all transgenic plants were highly sensitive to drought stress, OsCP3ox showed improved tolerance (5.0 – 5.3 score; rating scale from 1, highly tolerant to 9, highly sensitive) to salinity stress. Measurements of agronomic traits of the overexpression lines showed altered phenotype especially in plant height wherein OsCP2ox (123.8 – 132.7 cm) and OsCP5ox (117.9 – 119.6 cm) transgenic lines were significantly taller than the wild type Dongjin (109.8 cm), whereas OsCP3ox lines were significantly shorter (95.9 – 102.8 cm).

In chapter 3, sufficient transcript data were generated by employing de novo transcriptome profiling to investigate changes in gene expression during early response to X. oryzae pv. oryzae infection. Early interaction with the pathogen was inferred from the cDNA library of OsCP3ox-3 overexpression transgenic line infected with Xoo race K3a for 30 min representing incompatible interaction and from the cDNA library of infected OsCP3RNAi-56 transgenic line and Dongjin which represent compatible interaction. The total number of genes identified from the clean reads matching to the reference genome is 35,666. By implementing the log2FC≥1 and p<0.05 statistical thresholds, a total of 1,597 combined differentially expressed genes (DEGs) were identified in three libraries, 1,147 of which are exclusively regulated only in OsCP3ox-3, 111 in Dongjin, and 122 in OsCP3RNAi-56 sample. In all library samples, the frequency of activated genes is higher (925 in OsCP3ox-3, 194 in Dongjin, and 191 in OsCP3RNAi-56) than suppressed ones (511, 61, and 31, respectively). The 148 common genes between three samples were found to enrich (FDR<0.05) 27 biological process; among them high number of genes was assigned to metabolic process, primary metabolic process, response to stimulus, and response to stress. Enrichment analysis of unique set of DEGs in each library revealed more over-represented biological processes that are crucial in resistance response in OsCP3ox-3 library (n=50) over Dongjin (n=12) and OsCP3RNAi-56 (n=13) samples. Interestingly, defense response, response to stress, response to osmotic stress, response to hormone, ROS metabolic process, and signal transduction are exclusively identified only in OsCP3ox-3. A total of 548 OsCP3ox-3-specific genes are assigned into 35 Kyoto encyclopedia of genes and genomes (KEGG) pathways, 8 Dongjin-specific genes into 2 pathways, and 32 OsCP3RNAi-56-specific genes into 6 pathways. Mapman visualization of core biotic stress responsive genes in OsCP3ox-3 sample highlights signaling by receptor-like kinases (RLKs), calcium signals, G-proteins, and hormones, as well as transcription activity by members of ERF, bZIP, MYB and DOF as drastic early response upon infection which orchestrate downstream responses including activation of 13 putative pathogenesis-related (PR) proteins and 37 putative secondary metabolites-related
genes. Moreover, high H2O2 production and elevated accumulation of free SA are integral part of defense mechanism exhibited by OsCP3ox lines. Protein-protein interaction network analysis further depicted a more significant biological connection among proteins in OCP3ox-3 line with 11,696 (p<1.0e-16) predicted interactions over 132 (p=0.0152) and 167 (p=0.00213) interactions predicted in Dongjin and OsCP3RNAi-56, respectively.

In chapter 4, comparative transcriptional profile was analyzed in response to Xoo race K2 and K3a response in rice. The K2-DEG gene set was derived from the cDNA oligo microarray of RNA collected from 48-hour infected Jinbaek (representing intermediate to late incompatible interaction; R-K2) while the K3a-DEG gene set was generated using the RNA-sequencing of the 30 min post-inoculated RNA of OsCP3ox-3 overexpression transgenic line (representing early incompatible interaction, R-K3a). A huge gap in the magnitude of DEGs was found, wherein K3a, being the most recent race and more virulent induced more (n=1,355) DEGs than K2 (n=789). In both libraries, activated genes are more abundant which indicate extensive regulation of larger gene network that led to resistance. A total of 127 overlapping genes was identified, 43 of which enriched peroxidase activity (p=0.041), defense response (p=0.025), transcription factor activity (p=0.006), cell redox homeostasis (p=0.048), metal-binding (p=0.000), and secondary metabolites biosynthesis (p=0.033). Mapman provides an overview on extensive activation of signaling and transcription factor activity as conserved cellular response to both Xoo races. The number of activated genes was 45 in R-K2 and 37 in R-K3a for receptor kinase-signaling, 6 in R-K2 and 12 in R-K3a for calcium signaling, 3 in R-K2 and 3 in R-K3a for G-protein signaling, and 11 in R-K2 and 25 in R-K3a for hormone signaling. Activation of downstream mechanism in response to both races commonly rely
on WRKY, however K3a infection also requires substantial activation by ERF, MYB, and DOF, whereas K2-induced response also depends on MAPK signal transduction. It also appears that activation of multiple pathogenesis-related proteins were conserved cellular and physiological responses to K2 (n=11) and K3a (n=13) infection.
Interestingly, response to two races differs in terms of cell wall-related functions and lignin biosynthesis with K3a infection inducing 14 isoprenoids, 7 phenylpropanoids, 6 lignin, 1 wax, 8 flavonoids, and 2 alkaloid-like, while response to K2 only involves up-regulation of 6 terpenoids, 2 phenylpropanoids, and 2 flavonoids.
In summary, the analyses presented in this dissertation provide a detailed annotation of cysteine protease genes in rice. Using gain-of-function and RNAi-mediated knockdown of PLCP, I was able to demonstrate the functional aspect of this gene particularly against bacterial blight infection in rice. The analyses presented by RNA-sequencing, which is the first transcriptional investigation ever done on PLCPs in response to pathogen infection in rice, provide a global view of the transcriptome modulation response to Xoo mediated by cysteine protease. Substantial findings revealed that overexpression of cysteine protease allowed rice to circumvent Xoo infection through extensive activation of transduction signal and transcription that orchestrate downstream responses including up-regulation of multiple pathogenesis-related proteins and biosynthesis of secondary metabolites. Thus, rice PLCPs are valuable gene resource that can be employed in rice breeding programs for biotic stress.

목차 (Table of Contents)

• SUMMARY 1
• LITERATURE REVIEW 7
• LITERATURE CITED 25
• CHAPTER I. CHARACTERIZATION OF CYSTEINE PROTEASE GENE FAMILY IN RICE 37
• ABSTRACT 37
• INTRODUCTION 39
• MATERIALS AND METHODS 42
• Database searching for OsCP genes 42
• Gene structure and protein sequence analysis of OsCP genes 42
• Phylogenetic, motif, and protein structure analysis of OsCP genes 42
• Cis-element analysis in the promoters of the OsCP genes 43
• Stress treatment and RNA extraction 43
• cDNA library construction and qRT-PCR assay 44
• RESULTS 45
• Structural classification of cysteine proteases in rice 45
• Identification and phylogenetic analysis of putative OsCP genes 46
• Exon-intron structure and protein analysis of OsCP genes 47
• Motif and protein structure analysis of the OsCP genes 51
• Cis-element analysis of OsCP genes 56
• Selection of OsCP2, OsCP3, and OsCP5 56
• Expression analysis of OsCP genes 60
• DISCUSSION 64
• LITERATURE CITED 69
• CHAPTER II. OSCP GENES ENHANCED RESISTANCE AGAINST XANTHOMONAS ORYZAE PV. ORYZAE IN RICE 75
• ABSTRACT 75
• INTRODUCTION 77
• MATERIALS AND METHODS 80
• Isolation of Cysteine protease gene 80
• Overexpression vector construction 81
• RNAi vector construction 81
• Rice transformation 81
• DNA extraction and genomic PCR 83
• Identification of transgenic plants 84
• Pathogenicity test 84
• In planta bacterial growth analysis 84
• Abiotic stress screening 85
• Phenotypic evaluation in the field 86
• Statistical analysis 86
• RESULTS 87
• Molecular characterization of OsCP2, OsCP3, and OsCP5 87
• Development of transgenic rice with OsCP2, OsCP3, and OsCP5 87
• Evaluation of resistance against X. oryzae v. oryzae 91
• Abiotic stress screening of transgenic lines 93
• Agronomic traits of transgenic rice 95
• DISCUSSION 97
• LITERATURE CITED 101
• CHAPTER III. COMPARATIVE TRANSCRIPTOME ANALYSIS OF CYSTEINE-PROTEASE GENE AGAINST XANTHOMONAS ORYZAE PV. ORYZAE IN RICE 105
• ABSTRACT 105
• INTRODUCTION 107
• MATERIALS AND METHODS 110
• Identification of optimum treatment incubation period for gene expression 110
• Plant materials and treatment conditions 111
• RNA isolation 111
• RNA Sequencing 112
• Analysis of RNA sequencing reads and identification of differentially expressed genes 112
• Gene ontology analysis 113
• Quantification of aqueous hydrogen peroxide (H2O2) content 113
• Spectrophotometric analysis of salicylic acid in rice leaves 114
• RESULTS 115
• Analysis of RNA-seq libraries and fragment reads alignment 114
• Differentially expressed genes between wild type, OsCP3ox-3, and OsCP3RNAi-56 transgenic lines 116
• Identification of common and unique differentially expressed genes 119
• Functional enrichment analysis of overlapping genes 119
• Functional classification of DEGs unique to each sample library 121
• Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis 125
• Putative genes involved in plant hormone signal transduction pathway 125
• Putative core biotic responsive genes in OsCP3ox-3 transgenic sample 128
• Putative genes involved in secondary metabolism in OsCP3ox-3 sample 131
• Putative resistance and TAL-effector target genes identified in OsCP3ox-3 sample 131
• OsCP3ox-3 DEGs enrichment of reactive oxygen species (ROS) metabolic pathway 132
• Predicted protein-protein interaction network 134
• DISCUSSION.. 135
• LITERATURE CITED. 140
• CHAPTER IV. GENOME-WIDE TRANSCRIPTOME ANALYSIS OF RESPONSE AGAINST DIFFERENT RACES OF XANTHOMONAS ORYZAE PV. ORYZAE IN RICE.. 145
• ABSTRACT 145
• INTRODUCTION. 146
• MATERIALS AND METHODS 148
• Microarray and RNA sequencing experiments 148
• Validation of Xoo K2 and K3a-induced genes by qRT-PCR 149
• Comparative analysis of K2- and K3a-induced DEGs 150
• RESULTS 151
• Validation of the selected DEGs by qRT-PCR 151
• Differentially expressed genes in response to K2 and K3a 152
• Identification of putative conserved protein domain and features 153
• Common and contrasting transcriptional responses against Xoo K2 and K3a 154
• Comparative analysis of biotic stress-associated transcripts induced by K2 and K3a 154
• Predicted protein-protein interaction (PPI) networks 158
• DISCUSSION 160
• LITERATURE CITED 164