The relationship between cyclobutane pyrimidine dimer photolyase activity and disease resistance to the rice blast fungus Magnaporthe oryzae in African rice

概要

Ultraviolet B (UVB, 280–320 nm) sensitivity varied widely among Asian (Oryza sativa L.), and the activity of cyclobutane pyrimidine dimer (CPD) photolyase, which repairs UVB-induced CPDs, determine the UVB sensitivity 1. The UV-resistant rice has significantly increased CPD photorepair ability compared to UV-sensitive rice due to variation of CPD photolyase activity resulted from natural occurring polymorphisms within CPD photolyase gene 2–4, and thus the UVB sensitivity is highly correlated to CPD photolyase activity. In fact, UVB resistant CPD photolyase overexpress transgenic plants have been generated using Asian rice cultivars, with significant improved in UVB resistant than wild type plants 1,5, suggesting that CPD photolyase is crucial factors for determining the UVB sensitivity. UVB radiation can also induce defensive mechanisms and reduce oxidative damage 6, boost plant defense system against pathogenic fungi 7, such as H. parasitica 8 and Botrytis cinerera 9.

African rice (Oryza glaberrima Steud. and Oryza barthii A. Chev.) is a different species, that differs from O. sativa in many qualitative and quantitative traits such as differences in ligule shape, panicle branching, tiller number formation and growing phenotype; and it has independently domesticated in West Africa by possess traits for increased tolerance to biotic and abiotic stresses such as drought, soil acidity, iron and aluminium toxicity 10,11. Thus, one might expect that rice cultivated in tropical areas of Africa must have developed some UVB resistant mechanisms. However, the information about UVB sensitivity of African rice, generation of UVB resistant transgenic rice plant is largely absent, possibly due to challenge in transformation and regeneration of African local cultivar. Although African rice have shown to be resistant to several biotic stresses, the information about the interaction between UVB sensitivity and resistance to the rice blast fungus Magnaporthe oryzae have remained unexplored. Thus, the aim of this study was to understand the UVB sensitivity of African rice, generate of UVB resistant transgenic African rice, and examine the relationship between UVB sensitivity and resistance to the rice blast fungus M. oryzae in African rice.

Chapter 1 The UVB sensitivity is highly correlated with CPD photolyase activity 3,4. Although it grown in tropical environment with high UVB, the UVB sensitivity and CPD photolyase activity of African rice has remained largely unknown. In chapter I, I investigated the UVB sensitivity and CPD photolyase activity of 15 African rice cultivars. To achieve this purpose, 15 Africa cultivated rice including O. glaberrima, O. barthii and tropical O. sativa were grown under visible radiation with or without 1.2 W m-2 of supplementary UVB in growth chamber. Unexpectedly, most of African rice cultivars were more sensitive to UVB radiation (UVB super-hypersensitive) than even the hypersensitive Asian rice Surjamkhi 2 (Fig. 1A, B). The UVB super-hypersensitive cultivars have newly identified polymorphism of S78-R126-A283-Q296 in comparison to P78-Q126-G283-Q296 of UVB resistant Asian rice Sasanishiki (Fig. 1C). The activity of purified CPD photolyase of selected African rice (Fig. 1D, E) and total CPD photolyase activity (Fig. 1F), was significant lower in UVB super-hypersensitive and Surjamkhi compared to Sasanishiki and Norin 112. These results suggested that similar to Q to H296 on Surjamkhi2, polymorphisms of P to S78 and G to A283 largely reduces the CPD photorepair activity of UVB super-hypersensitive cultivars, and enhancing CPD photolyase activity can improve the UVB resistance of African rice.

Chapter 2 Overexpress CPD photolyase in transgenic plants have shown to improve the UVB resistant of Asian rice cultivars1,5. However, at current moment, to the best of my knowledge, there are no any reports that has generated the UVB tolerant transgenic African rice using TOG12380 (O. glaberrima) as parental line (PL) plant, possibly due to the difficulties in the transformation and regeneration of African local cultivars. Therefore, in chapter 2, I investigated about the creation of UVB resistance transgenic African rice cultivar. To achieve this purpose, the CPD photolyase gene of UVB resistant Asian rice Sasanishiki was ubiquitously expressed by the CaMV 35S promoter using UVB sensitive TOG12380 (O. glaberrima) as PL plant (Fig. 2A). The transgenic overexpress African rice (TOG12380-OxPHR) have significant improved in UVB resistance (Fig. 2B), with higher transcripts (Fig. 2C) and activity (Fig. 2D) of CPD photolyase than PL plants. These results strongly suggest that despite of species barrier, UVB-induced CPDs is still the main causes for UVB-induced growth hindrance in higher plants grown under supplementary UVB radiation stress, and CPD photolyase is proper bioengineering tools for developing the UVB resistant African rice.

Chapter 3 UVB radiation can induce defensive mechanisms and reduce oxidative damage 6, boost plant defense system against pathogenic fungi 7, such as H. parasitica 8 and Botrytis cinerera 9, both in Arabidopsis. The fact that UVB-hypersensitive (Surjamkhi) and super-hypersensitive (TOB7307) cultivars have been domesticated and are still cultivated today in tropical area of Bengal 13,14 and Africa 12, respectively, prompt me to ask whether such a high UVB sensitivity may be beneficial for surviving other stresses in tropical areas as pathogens. However, the relationship between CPD photolyase activity and resistance mechanisms to pathogens is poor understood. Thus, in chapters 3, I investigated the relationship between CPD photolyase activity and resistance to the rice blast fungus M. oryzae. To achieve this purpose, M. oryzae basal resistance was analysed by punch and leaf sheath assay on transgenic plants with different CPD photolyase activity (S-C line); Sasanishiki CDP photolyase overexpressed and (AS line); antisense line with high and low CPD photolyase activity 1, respectively, and TOG12380-OxPHR and TOG12380- WT, with high and low CPD photolyase activity, respectively. I discovered that 12 hours of 0.4 W m -2 UVB pretreatment, reduces secondary infection hyphae of AS line compared to S-C and Sasanishiki (Fig. 3A, B). Interestingly, UVB pretreatment enhances susceptibility to rice blast on the UVB resistant transgenic lines S-C (Fig. 3C) and TOG12380-OxPHR (Fig. 3D), with high CPD photolyase activity. Moreover, UVB pretreatment did not activates expression of OgJiPR10, OgWRKY45 and CAD 6 PR genes of TOG12380- OxPHR plants (Fig. 3E). Thus, similar to UVC-induced DNA damage that activate immune response prior to H. parasitica molecules recognition by plant 8, these results suggested that the high UVB-induced DNA damage in AS line and TOG12380-WT, due to low CPD photolyase activity, activates expression of PR genes and enhances basal resistance of M. oryzae prior to infection occurrence (Fig. 3C, D and E).

Conclusion In this study, I showed that African rice cultivars are highly sensitive to UVB radiation (UVB super-hypersensitive) than even the hypersensitive Asian rice Surjamkhi, due to polymorphism of S78-R126- A283-Q296 in comparison to P78-Q126-G283-Q296 of UVB resistant Asian rice Sasanishiki. The transgenic rice African rice plant generated here, TOG12380-OxPHR, have significant improved in UVB resistance compared with WT plant due to high CPD photolyase activity. On the other hand, UVB pretreatment on S- C line and TOG12380-OxPHR, enhances M. oryzae susceptibility, and less activation of PR genes. Thus, the low CPD photolyase activity of Surjamkhi and TOB7307 could be one of the beneficial adaptation strategies for combating the most dangerous stresses in tropical, such as pathogens, as shown here on rice blast fungus M. oryzae (Fig. 3F). This study offers knowledge for development of UVB and M. oryzae resistant rice, with high yield and productivity.

この論文で使われている画像