The OsOXO2, OsOXO3 and OsOXO4 Positively Regulate Panicle Blast Resistance in Rice

Abstract

Background: Although panicle blast is more destructive to yield loss than leaf blast in rice, the cloned genes that function in panicle blast resistance are still very limited and the molecular mechanisms underlying panicle blast resistance remain largely unknown.

Results: In the present study, we have confirmed that the three Oxalate oxidase (OXO) genes, OsOXO2, OsOXO3 and OsOXO4 from a blast-resistant cultivar BC10 function in panicle blast resistance in rice. The expression of OsOXO2, OsOXO3 and OsOXO4 were induced by panicle blast inoculation. Subcellular localization analysis revealed that the three OXO proteins are all localized in the nucleus and cytoplasm. Simultaneous silencing of OsOXO2, OsOXO3 and OsOXO4 decreased rice resistance to panicle blast, whereas the OsOXO2, OsOXO3 and OsOXO4 overexpression rice plants individually showed enhanced panicle blast resistance. More H₂O₂ and higher expression levels of PR genes were observed in the overexpressing plants than in the control plants, while the silencing plants exhibited less H₂O₂ and lower expression levels of PR genes compared to the control plants. Moreover, phytohormone treatment and the phytohormone signaling related gene expression analysis showed that panicle blast resistance mediated by the three OXO genes was associated with the activation of JA and ABA signaling pathways but suppression of SA signaling pathway.

Conclusion: OsOXO2, OsOXO3 and OsOXO4 positively regulate panicle blast resistance in rice. The OXO genes could modulate the accumulation of H₂O₂ and expression levels of PR gene in plants. Moreover, the OXO genes mediated panicle blast resistance could be regulated by ABA, SA and JA, and may be associated with the activation of JA and ABA signaling pathways but suppression of the SA signaling pathway.

Keywords: OXO (oxalate oxidase); Panicle blast; Rice (Oryza sativa L.).

Fig. 1

The temporal and spatial expression patterns of OsOXO2, OsOXO3 and OsOXO4 and their subcellular localizations. A. The expression of OsOXO2, OsOXO3 and OsOXO4 were assessed by quantitative RT-PCR at 6 h, 12 h, 24 h, 48 h after panicle blast inoculation. Error bars indicate the standard deviation (SD) from three biological replicates and ** indicates a statistically significant difference compared with mock (t test, P < 0.01). B. Sub-cellular localization of OsOXO2, OsOXO3 and OsOXO4 in rice protoplasts. Bar = 10 μm. C. Expression analysis of OsOXO2, OsOXO3 and OsOXO4 in different rice tissues by quantitative RT-PCR and GUS activity of OsOXO4 in the panicles of Nipponbare. Error bars indicate the standard deviation (SD) from three biological replicates. a, panicle at the early booting stage; b, panicle at the booting stage; c, panicle at the initial heading stage

Fig. 2

The disease phenotypes of the OXO gene overexpressing plants before and during panicle blast infection. OEOXO2 indicates the plants overexpressing OsOXO2, OEOXO3 indicates the plants overexpressing OsOXO3 and OEOXO4 indicates the plants overexpressing OsOXO4. a Transcription analysis of OsOXO2, OsOXO3 and OsOXO4 in their corresponding overexpressing plants. Error bars indicate the SD from three biological replicates. ** indicates a statistically significant difference compared with PHQSN (t test, P < 0.01). b Disease phenotypes of OEOXO2, OEOXO3, OEOXO4, and the empty vector control (PHQSN) plants at the heading stage after inoculation with M. oryzae using the cotton-wrapping method. c Correlation analysis between enhanced resistance and the expression levels of OsOXO2, OsOXO3 and OsOXO4 in the overexpressing plants after panicle blast inoculation. The correlation coefficient is calculated by linear regression

Fig. 3

The disease phenotypes of OXO silencing (RNAi) plants after panicle blast infection. a Transcription analysis of OsOXO2, OsOXO3 and OsOXO4 in the RNAi plants by quantitative RT-PCR.Error bars indicate the standard deviation (SD) from three biological replicates and asterisks indicate statistically significant differences compared with ck (PHQSN) (t test, **P < 0.01, *P < 0.05). b Disease phenotypes of the RNAi and control (PHQSN) plants at the heading stage after inoculation with M. oryzae using the cotton-wrapping method. NI indicates the non-inoculation panicle. c Correlation analysis between decreased resistance and the expression levels of OsOXO3 and OsOXO4 in the RNAi plants after panicle blast inoculation. The correlation coefficient is calculated by linear regression

Fig. 4

Expression analysis of pathogenesis-related (PR) genes in the OXO transgenic plants and control plants by quantitative RT-PCR. Error bars indicate the SD from three biological replicates and asterisks indicate statistically significant differences compared to the control plants (t test, **P < 0.01; *P < 0.05)

Fig. 5

Hydrogen peroxide (H₂O₂) contents in the OXO transgenic plants and control plants. a H₂O₂ contents in the panicles of PHQSN, OEOXO2, OEOXO3, OEOXO4 and RNAi plants. b DAB staining of the leaves for PHQSN, OEOXO2, OEOXO3, OEOXO4 and RNAi plants. Error bars indicate the SD of at least ten biological replicates and ** indicates significant differences between the transgenic and control plants (t test, P < 0.01). FW means fresh weight

Fig. 6

Time-course transcription analysis of OsOXO3 and OsOXO4 in leaves after abscisic acid (ABA), ethylene (ET), salicylic acid (SA), jasmonic acid (JA) treatments by quantitative RT-PCR. Error bars indicate the SD from three biological replicates and asterisks indicate statistically significant differences compared with water treatment (t test, **P < 0.01 and *P < 0.05)

Fig. 7

The expression of plant hormone related genes in panicles of the OXO transgenic plants and control plants by quantitative RT-PCR. Error bars indicate the SD from three biological replicates and asterisks indicate statistically significant differences compared to the control plants (t test, **P < 0.01 and *P < 0.05)