Understanding Host-Pathogen Interactions with Expression Profiling of NILs Carrying Rice-Blast Resistance Pi9 Gene

Abstract

Magnaporthe oryzae infection causes rice blast, a destructive disease that is responsible for considerable decrease in rice yield. Development of resistant varieties via introgressing resistance genes with marker-assisted breeding can eliminate pesticide use and minimize crop losses. Here, resistant near-isogenic line (NIL) of Pusa Basmati-1(PB1) carrying broad spectrum rice blast resistance gene Pi9 was used to investigate Pi9-mediated resistance response. Infected and uninfected resistant NIL and susceptible control line were subjected to RNA-Seq. With the exception of one gene (Pi9), transcriptional signatures between the two lines were alike, reflecting basal similarities in their profiles. Resistant and susceptible lines possessed 1043 (727 up-regulated and 316 down-regulated) and 568 (341 up-regulated and 227 down-regulated) unique and significant differentially expressed loci (SDEL), respectively. Pathway analysis revealed higher transcriptional activation of kinases, WRKY, MYB, and ERF transcription factors, JA-ET hormones, chitinases, glycosyl hydrolases, lipid biosynthesis, pathogenesis and secondary metabolism related genes in resistant NIL than susceptible line. Singular enrichment analysis demonstrated that blast resistant NIL is significantly enriched with genes for primary and secondary metabolism, response to biotic stimulus and transcriptional regulation. The co-expression network showed proteins of genes in response to biotic stimulus interacted in a manner unique to resistant NIL upon M. oryzae infection. These data suggest that Pi9 modulates genome-wide transcriptional regulation in resistant NIL but not in susceptible PB1. We successfully used transcriptome profiling to understand the molecular basis of Pi9-mediated resistance mechanisms, identified potential candidate genes involved in early pathogen response and revealed the sophisticated transcriptional reprogramming during rice-M. oryzae interactions.

Keywords: Magnaporthe oryzae; RNA-Seq; WRKY; ethylene; host-pathogen interaction; jasmonic acid; near isogenic lines; rice blast.

Figure 1

Disease interaction and expression profile of resistant (PB1+Pi9) and susceptible (PB1) lines 24 hpi with Magnaporthae oryzae. (A) a: M. oryzae spore, b: compatible reaction of PB1 line, and c: Incompatible reaction of PB1+Pi9 NIL. (B) Expressed loci common between PB1+Pi9 and PB1 with M. oryzae inoculation and without M. oryzae inoculation (mock). (C) Significant (FDR adjusted p ≤ 0.05) differentially expressed loci (log₂ fold change ≥2) common across PB1+Pi9 and PB1. (D) Heat map of significant (FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) differentially expressed loci of PB1+Pi9 and their respective log₂ fold change in PB1. Red represents up-regulated loci and green represents down-regulated loci.

Figure 2

Magnaporthe infection and RNA-Seq workflow of PB1+Pi9 and PB1.

Figure 3

MapMan overview of significant (FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) differentially expressed loci (SDEL) involved in biotic stress pathway, unique to PB1+Pi9, upon M. oryzae infection. SDEL are binned to MapMan functional categories and values represented as log₂ fold change values. Red represents up-regulated loci and green represents down-regulated loci. JA, jasmonic acid; SA, salicylic acid; bZIP, basic region-leucine zipper; ERF, ethylene response factor; MAPK, mitogen-activated protein kinase; PR-protein, pathogenesis-related protein.

Figure 4

The qRT-PCR validation of significant differentially expressed loci (SDEL) in PB1+Pi9 upon M. oryzae infection. Blue bar represents the absolute fold change of PB1+Pi9. Actin was used for transcript normalization. Standard error bar show the standard deviation for three triplicate assay. Abbreviations: WRKY71, WRKY71 transcription factor (LOC_Os02g08440); WRKY76, WRKY76 transcription factor (LOC_Os09g25060); WRKY108, WRKY108 transcription factor (LOC_Os01g60600); bZIP, bZIP transcription factor domain (LOC_Os07g48660); TF HBP-1b, transcription factor HBP-1b (LOC_Os01g06560); AP2, AP2 domain containing protein (LOC_Os02g45420); POX, Peroxidase (LOC_Os08g02110); TRX, thioredoxin (LOC_Os07g29410); DR, disease resistance protein SlVe2 precursor (LOC_Os01g06836); NB/LRR DR, NB-ARC/LRR disease resistance protein (LOC_Os04g43440); PR, pathogenesis-related Bet v I (LOC_Os08g28670); rp3, rp3 protein (LOC_Os12g03080); Cf-2, Cf-2 (LOC_Os01g06876); CHIT, CHIT1-Chitinase (LOC_Os02g39330); OsWAK, OsWAK95- OsWAK receptor-like protein kinase (LOC_Os10g02250); CYP450, cytochrome P450 (LOC_Os06g39780).

Figure 5

Metabolism overview of significant differentially expressed loci (FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) unique to PB1+Pi9. Metabolism overview covers the primary and secondary metabolism, upon M. oryzae infection in PB1+Pi9. SDEL are binned to MapMan functional categories and values represented as log2-transformed values. Red represents up-regulated loci and green represents down-regulated loci.

Figure 6

Number of significant differentially expressed loci (SDEL; FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) unique to PB1+Pi9 present in different biological processes and molecular functions of all significant GO terms. GO terms for biological processes are response to stimulus (GO:0050896), response to stress (GO:0006950), secondary metabolic process (GO:0019748), response to endogenous stimulus (GO:0009719), response to biotic stimulus (GO:0009607), lipid metabolic process (GO:0006629), photosynthesis (GO:0015979) with p-value of 1.00E-08, 1.30E-06, 0.00067, 0.002, 0.0099, 0.025, 0.025, 0.056, respectively. GO terms of molecular function are oxygen binding (GO:0019825), transcription regulator activity (GO:0030528), transcription factor activity (GO:0003700) and catalytic activity (GO:0003824) with p-value of 0.00027, 0.0014, 0.0014, 0.016, respectively.

Figure 7

Heat-map of significant differentially expressed loci (FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) unique to PB1+Pi9 present in the gene ontology term, response to biotic stimulus (GO:0009607) and their respective log₂ fold change in both PB1+Pi9 and PB1. Red represents up-regulated loci and green represents down-regulated loci.

Figure 8

Co-expression network of proteins corresponding to significant differentially expressed loci (SDEL; FDR adjusted p ≤ 0.05 & log₂ fold change ≥2) unique to PB1+Pi9 in response to biotic stimulus. In the network, circular node represents the protein encoded by the respective SDEL and the interconnecting lines represent the source by which protein interactions are derived. The protein interactions are derived from coexpression, concurrence, experiments, neighborhood, database, text mining, and homology sources. All the interactions represented have score greater than 0.4 that shows medium confidence for the network.

Figure 9

A model representing Pi9-mediated resistance upon M. oryzae infection in PB1+Pi9. Upon germination, M. oryzae spores penetrate the leaf cuticle, where upon fungal effectors are recognized by the R (Pi9) protein. In turn, Pi9 triggers ROS production and activating signaling molecules (kinases and hormones). Signaling molecules then activate transcription factors (MYB, WRKY, bZIP) that trigger the expression of defense response genes (Pox, Pal, Lac, Chit, Glc, Lpo), leading to the synthesis of antimicrobial compounds (PHY, PHO) that curtails M. oryzae growth. Inverted red triangle represents effector proteins secreted by germinating M. oryzae spores invading cell wall. Abbreviations: ROS, reactive oxygen species; HR, hypersensitive response; PCD, programmed cell death; TF, transcription factor; ET, ethylene; JA, jasmonic Acid; PR proteins, pathogenesis-related proteins; CA, cinnamic acid; PAL, phenylalanine ammonia lyase; Glc, glucanases; Chit, chitinases; LPO, lipoxygenases; POX, peroxidases; Lac, laccases; PHO, pheolics; PHY, phytoalexin.