OsWRKY67 positively regulates blast and bacteria blight resistance by direct activation of PR genes in rice

Abstract

Background: WRKY proteins are one of the largest gene families and are well-known for their regulatory roles in many aspects of plant development, including plant response to both biotic and abiotic stresses. Although the roles of WRKY proteins in leaf blast resistance have been well-documented in rice, their functions in panicle blast, the most destructive type of blast disease, are still largely unknown.

Results: Here, we identified that the transcription of OsWRKY67 was strongly activated by leaf and panicle blast infection. OsWRKY67 is ubiquitously expressed and sub-localized in the nucleus. Rice plants overexpressing OsWRKY67 showed quantitatively enhanced resistance to leaf blast, panicle blast and bacterial blight. In contrast, silencing of OsWRKY67 increased the susceptibility to blast and bacterial blight diseases. RNA-seq analysis indicated that OsWRKY67 induces the transcription of a set of defense-related genes including the ones involved in the salicylic acid (SA)-dependent pathway. Consistent with this, the OsWRKY67-overexpressing plants accumulated higher amounts of endogenous SA, whereas lower endogenous SA levels were observed in OsWRKY67-silenced plants relative to wild-type Nipponbare plants before and after pathogen attack. Moreover, we also observed that OsWRKY67 directly binds to the promoters of PR1a and PR10 to activate their expression.

Conclusions: These results together suggest the positive role of OsWRKY67 in regulating rice responses to leaf blast, panicle blast and bacterial blight disease. Furthermore, conferring resistance to two major diseases makes it a good target of molecular breeding for crop improvement in rice.

Keywords: Bacterial blight; Leaf blast; OsWRKY67; Panicle blast; Salicylic acid.

Fig. 1

The expression pattern of OsWRKY67 after blast fungus infection. The values are means ± standard deviations (SDs) of three biological replicates, and asterisks represent significant differences relative to the water treatment (0 h) (t-test, **P < 0.01 and *P < 0.05). a The transcription of OsWRKY67 was activated by leaf blast infection. LB = leaf blast. b The transcription of OsWRKY67 was activated by panicle blast infection. PB = panicle blast

Fig. 2

The spatiotemporal expression of OsWRKY67 in Nipponbare plants. a Relative expression levels of OsWRKY67 in different Nipponbare tissues. Young leaf, leaf at the three- to four-leaf stage; old leaf, leaf at the booting stage. The values are means ±SDs of three biological replicates. b-j GUS staining analysis of OsWRKY67 in different Nipponbare tissues. b leaf at the booting stage; c sprouting stage; d hull; e one-week old seedling; f root at the booting stage; g the second node at the booting stage; h the first node at the booting stage; i panicle at the booting stage; j panicle at the heading stage

Fig. 3

Phenotypes of the OsWRKY67 overexpressing (OX-WRKY67) plants infected with blast fungus. The asterisks represent significant differences relative to Nipponbare plants (t-test, **P < 0.01). a Relative transcription levels of OsWRKY67 in wild-type Nipponbare (NPB) and OX-WRKY67 plants. The values are mean ± SD of three biological replicates. b The OX-WRKY67 plants exhibited enhanced leaf blast resistance after inoculation with GD08-T13 using the punch method. c Relative lesion size in Nipponbare and OX-WRKY67 plants after infection by leaf blast. The values are mean ± SD of twelve biological replicates. d Numbers of spores produced on the Nipponbare and OX-WRKY67 plants after punch inoculation. The values are means ± SDs of six biological replicates. e The OX-WRKY67 plants exhibited enhanced panicle blast resistance after inoculation with GD08-T13. f The infected main axis length in Nipponbare and OX-WRKY67 plants after infection by panicle blast. The values are means ± SDs of twenty biological replicates

Fig. 4

Phenotypes of the OsWRKY67-silenced (WRKY67-RNAi) plants infected with blast fungus. The asterisks represent significant differences relative to wild-type Nipponbare (NPB) plants (t-test, **P < 0.01 and *P < 0.05). a Relative transcription levels of OsWRKY67 in Nipponbare and WRKY67-RNAi plants. The values are means ± SDs of three biological replicates. b Silencing of OsWRKY67 increased susceptibility to leaf blast infection using punch method. c Relative lesion size in Nipponbare and WRKY67-RNAi plants after leaf blast infection. The values are means ± SDs of twelve biological replicates. d Numbers of spores produced on Nipponbare and WRKY67-RNAi plants after punch inoculation. The values are means ± SDs of six biological replicates. e Silencing of OsWRKY67 increased susceptibility to panicle blast in rice. f The infected main axis length in Nipponbare and WRKY67-RNAi plants after panicle blast inoculation. The values are means ± SDs of twelve biological replicates

Fig. 5

OsWRKY67 regulates the transcription of genes involved in the salicylic acid (SA) signaling pathway. The wild-type Nipponbare and transgenic plants were inoculated with GD08-T13 at the three- to four-leaf stage. The values are means ± SDs of three biological replicates and the asterisks represent significant differences relative to Nipponbare plants at **P < 0.01 or *P < 0.05 by t-tests. The transcript level of Nipponbare plants was set to “1” at each time point

Fig. 6

OsWRKY67 confers disease resistance in a SA-dependent manner. The values are means ± SDs of three biological replicates and the asterisks represent significant differences relative to the water treatment (0 h) or Nipponbare plants at **P < 0.01 and *P < 0.05 by t-test. a The expression of OsWRKY67 was activated by exogenous SA. CK = water treatment. b Transcriptionally modulating OsWRKY67 influenced the accumulation of SA

Fig. 7

OsWRKY67 binds specifically to the W-box elements. a Yeast one-hybrid assays indicated that OsWRKY67 can bind directly to the sequences of W-box (BS65) and its own promoter (67p). P53 is the positive control. NC is the negative control which did not contain the W-box and mBS65 is the mutated probe of BS65 with TGAC sequence changed to CCTA. The detailed sequences of BS65 and mBS65 are shown in section c. b Yeast one-hybrid assays indicated that OsWRKY67 can bind directly to the promoters of PR1a and PR10 through the W-box motif. NC is the negative control from the PR10 promoter which did not contain the W-box motif. c The EMSA experiment showed the binding of OsWRKY67 protein to the sequences of the W-box. An excess of unlabeled-BS65 probe was added as competitors. The negative control was conducted by inoculating the Biotin-labeled BS65 probe without the OsWRKY67-His protein. d The EMSA experiment showed the binding of OsWRKY67 protein to its own promoter through the W-box element. The oligonucleotides 67p and m67p were used as the probes. An excess of unlabeled-67p probe was added as competitors. e The EMSA experiment showed the binding of OsWRKY67 protein to the promoter of PR10. The oligonucleotides P10 and mP10 were used as the probes. An excess of unlabeled-P10 probe was added as competitors. f The EMSA experiment showed the binding of OsWRKY67 protein to the promoter of PR1a. The oligonucleotides P1 and mP1 were used as the probes. An excess of unlabeled-P1 probe was added as competitors