OsWRKY22, a monocot WRKY gene, plays a role in the resistance response to blast

Abstract

With the aim of identifying novel regulators of host and nonhost resistance to fungi in rice, we carried out a systematic mutant screen of mutagenized lines. Two mutant wrky22 knockout lines revealed clear-cut enhanced susceptibility to both virulent and avirulent Magnaporthe oryzae strains and altered cellular responses to nonhost Magnaporthe grisea and Blumeria graminis fungi. In addition, the analysis of the pathogen responses of 24 overexpressor OsWRKY22 lines revealed enhanced resistance phenotypes on infection with virulent M. oryzae strain, confirming that OsWRKY22 is involved in rice resistance to blast. Bioinformatic analyses determined that the OsWRKY22 gene belongs to a well-defined cluster of monocot-specific WRKYs. The co-regulatory analysis revealed no significant co-regulation of OsWRKY22 with a representative panel of OsWRKYs, supporting its unique role in a series of transcriptional responses. In contrast, inquiring a subset of biotic stress-related Affymetrix data, a large number of resistance and defence-related genes were found to be putatively co-expressed with OsWRKY22. Taken together, all gathered experimental evidence places the monocot-specific OsWRKY22 gene at the convergence point of signal transduction circuits in response to both host and nonhost fungi encountering rice plants.

Figure 1

Phenotypes of the two wrky22 T‐DNA insertion lines and corresponding nullizygous (wild‐type, Wt) plants after Magnaporthe infection. (a) Symptoms of 4‐week‐old wrky22 mutant lines upon M. oryzae virulent FR13 infection at 7 days post‐inoculation. (b) Symptoms of 2‐week‐old wrky22 mutant lines upon M. oryzae avirulent CL3.6.7 infection. (c) Number of grey lesions surrounded by brown zones in wrky22 mutants and corresponding nullizygous (Wt) plants at 7 days post‐inoculation with the avirulent M. oryzae strain CL3.6.7. (d) Nested reverse transcription‐polymerase chain reaction (RT‐PCR) of OsWRKY22 and house‐keeping actin expression in wrky22 mutants and Wt plants before inoculation.

Figure 2

Magnaporthe oryzae, M. grisea and Blumeria graminis f. sp. hordei (Bgh) leaf infection process at the cellular level in wrky22 mutants and corresponding nullizygous wild‐type (Wt) plants at 24 and 48 h post‐inoculation (hpi). (a) Classification of cytological interaction types revealed after 3,3′‐diaminobenzidine (DAB) staining (H₂O₂ production): I, appressorium with no brown cell; II, single brown cell; III, multiple brown cells. (b) Classification of cytological interaction types revealed after aniline blue staining (callose deposition): I, appressorium with no fluorescence; II, fluorescence under appressorium; III, fluorescence on part of the cell wall; IV, fluorescence on the whole cell wall; V, fluorescence inside the cell; VI, fluorescence occurring in the neighbouring cells. Left panels show Magnaporthe and right panels Bgh infection process. The histograms represent the frequency of DAB‐associated cellular phenotypes or callose deposition at 24 and 48 hpi with Magnaporthe FR13 (virulent), BR32 and CL3.6.7 (avirulent) isolates, the nonhost M. grisea BR29 and Bgh strains. The mean and standard deviation of three replicates (constituting 100 interactions) are shown (*P < 0.05). Bars, 20 µm.

Figure 3

Over‐expressing (OE) OsWRKY22 rice cv. Nipponbare T0 plants. (a) OsWRKY22 expression levels in OE transgenic lines and in empty vector control plants before inoculation. #1, #2 and #3 indicate the three selected T0 lines to obtain T1 progeny plants segregating for the introduced T‐DNA. (b) Enhanced disease resistance phenotype of the OE lines and phenotype of the empty vector plants upon M. oryzae FR13 virulent infection. A representative sample of leaves from OE and empty vector control plants at 7 days post‐inoculation is shown.

Figure 4

Over‐expressing (OE) OsWRKY22 rice cv. Nipponbare T1 plants. Three independent transgenic lines (#1, #2 and #3, all carrying only one T‐DNA) with 13, 26 and 27 plants for each line were analysed for phenotyping and molecular analysis. (a) Symptoms of three OE transgenic lines on Magnaporthe oryzae FR13 virulent infection: S, susceptible; R, resistant; PR, partially resistant. A representative sample of leaves from OE plants is shown. (b) Frequency of observed and expected S/R/PR phenotypes of OE transgenic lines and nullizygous (wild‐type, Wt) plants upon infection with FR13. (c) Basal OsWRKY22 expression levels in OE transgenic lines and nullizygous Wt plants before inoculation. The mean and standard deviation of three replicates are shown (*P < 0.05).

Figure 5

OsWRKY22 gene expression following Magnaporthe or mock inoculation by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The strains used were Magnaporthe FR13 (virulent) and CL3.6.7 (avirulent) isolates. Values represent the means of log₂(relative expression) and standard deviation for four biological replicates. Statistical differences between mock and infected samples were assessed by t‐test analysis (*P < 0.05). All calculations for relative quantification were performed as described in Pfaffl (2001) and the reference gene used was actin (Os03g50885).

Figure 6

In silico analysis of the monocot‐specific OsWRKY proteins. (a) Hierarchical clustering tree of the OsWRKYs obtained using the full protein sequences. Subgroups 3I, 3II and 3III are the three subclusters within group 3 identified in this study. (b) Neighbour‐joining tree based on all‐against‐all blastp best hits (E‐values > 1e⁻³⁰) of all group 3 OsWRKYs. The 19 WRKYs belonging to subgroups 3I and 3II (green dots) are identifiable within the monocot‐specific group (in green) bearing only sequences from cereals. Gm, Glycine max; Hv, Hordeum vulgare; Lj, Lotus japonica; Mt, Medicago truncatula; Pt, Populus trichocarpa; Rc, Ricinus communis; Sb, Sorghum bicolor; Ta, Triticum aestivum; Vv, Vitis vinifera; Zm, Zea mays. Outgroups: Ac, Areca catechu; Ma, Musa acuminate; Pa, Picea abies; Pp, Physcomitrella patens.

Figure 7

Co‐regulatory networks of the monocot/cereal‐specific (MCS) OsWRKY genes and other members of the OsWRKY family. (a) Linear Pearson analysis. (b) Logarithmic Pearson analysis. The 14 MCS OsWRKY genes (black nodes) and their best co‐regulated OsWRKYs (white nodes) are included in the graphical representation (lines connecting nodes represent Pearson coefficient threshold > 0.8).

Figure 8

Bar charts of the genes co‐expressed with OsWRKY22 putatively involved in the rice defence response (147 genes) according to their known or predicted function. The number of genes is shown on the x‐axis and the main categories on the y‐axis. LRR: different categories of leucine‐rich repeat (LRR) resistance genes; DRG: defence‐related genes; RR: rust resistance; Mla: MLA protein; BR: blight resistance. LTP: LTP family; Def: defensins; Sub: subtilisins; GLP: germin‐like proteins; Bp: BURP domain. Ext: extensin family; Flav: flavonoids‐related genes; Ank: ankyrin repeat family; Ter: terpene synthases putative; Lec: lectin domain; Ha: harpin‐induced protein. Sam: SAM‐dependent carboxyl methyltransferase; Cls: cellulose synthase‐like family. BTB: bric‐a‐brac tramtrack broad complex; P450: cytochrome P450; Ubi: several proteins belonging to the ubiquitin complex; Prot: proteases; Spk: speckle‐type POZ proteins; Ch: chorismate mutase. Kin: different categories of receptor kinase; Lip: GDSL‐like lipases; Cal: calmodulin‐binding proteins; R1: RGA‐1. Et: 1‐aminocyclopropane‐1‐carboxylate oxidase; Ja: jasmonate O‐methyltransferase. MYB: MYB transcription factor; HLH: helix–loop–helix DNA‐binding domain; Zn: zinc finger C3HC4‐type domain; Zp: bZIP transcription factor domain. F‐box: F‐box domain; Fmo: flavin mono‐oxygenase; Sn: SNARE domain; Ro: reticuline oxidase‐like protein.