Engineering durable nonspecific resistance to phytopathogens is one of the ultimate goals of plant breeding. However, most attempts to reach this goal fail as a result of rapid changes in pathogen populations and the sheer diversity of pathogen infection mechanisms. In this study, we show that the expression of a harpin-encoding gene (hrf1), derived from Xanthomonas oryzae pv. oryzae, confers nonspecific resistance in rice to the blast fungus Magnaporthe grisea. Transgenic plants and their T1–T7 progenies were highly resistant to all major M. grisea races in rice-growing areas along the Yangtze River, China. The expression of defence-related genes was activated in resistant transgenic plants, and the formation of melanized appressoria, which is essential for foliar infection, was inhibited on plant leaves. These results suggest that harpins may offer new opportunities for generating broad-spectrum disease resistance in other crops.
Rice blast causes between 10% and 30% yield losses in rice, posing a constant threat to the supply of the staple food for nearly one-half of the world's population (Zhu Y.Y. et al., 2000; Talbot, 2003). The control of Magnaporthe grisea relies on the use of resistant cultivars and the application of fungicides, but neither approach is particularly effective. Host resistance in rice to M. grisea functions via a classic gene-for-gene interaction in which a single dominant resistance gene corresponds with a dominant avirulence gene in the pathogen (Hammond-Kosack and Jones, 1997; Talbot, 2003). Because of apparent instability in the genome of M. grisea, new pathogenic races evolve rapidly, and thus host resistance typically only lasts for a few years (Zhu Y.Y. et al., 2000; Talbot, 2003). Few fungicides are available for the effective control of rice blast, as rapid mutation in the pathogen leads to the emergence of fungicide-resistant variants (Takagaki et al., 2004), and high-dose applications of fungicides pose risks to both humans and the environment. The generation of cultivars that possess nonspecific resistance to M. grisea would provide an economically effective and environmentally sound approach to rice blast control.
One promising approach to the achievement of nonspecific resistance to M. grisea is to incorporate genes that elicit general defence responses into rice (Dangl and Jones, 2001; Stuiver and Custers, 2001). Exogenous applications of harpins, protein elicitors isolated from plant pathogenic bacteria (Wei et al., 1992), induce systemic acquired resistance (SAR) in plants to pathogens by the activation of defence pathways mediated by salicylic acid (SA) (He et al., 1993; Dong et al., 1999), jasmonic acid (JA) or ethylene (Kariola et al., 2003). In addition, they induce a range of pathogenesis-related (PR) genes (Lee et al., 2001; Peng et al., 2004). The transformation of harpin-encoding (hrp) genes into rice has the potential to enhance general resistance to pathogens. In this study, we demonstrate that the expression of a harpin-encoding gene (hrf1), derived from Xanthomonas oryzae pv. oryzae, enhances the expression levels of defence-related genes, increases the silicon content of leaves, and interferes with the formation of appressoria. hrf1-transformed plants and their progenies were highly resistant to all major M. grisea races in rice-growing areas along the Yangtze River, China, an area with arguably the highest diversity of the pathogen in the world.
Rice transformation, and genetic and phenotypic analyses of hrf1-transformed rice plants
Rice (Oryza sativa ssp. Japonica) was transformed with the harpinXoo (hrf1) gene derived from X. oryzae pv. oryzae under the control of the 35S promoter (Zhu W.G. et al., 2000; Wen et al., 2003). Rice cultivar, R109, susceptible to rice blast, was transformed with hrf1-containing pBMH9 by Agrobacterium-mediated transformation (Figure S1, see ‘Supplementary material’) (Sambrook et al., 1989; Huang et al., 2001), producing 29 primary transgenic plants (T0). These plants were self-pollinated to produce T1 lines. Two independently derived T0 primary transgenic plants (NJH12 and NJH16) showed strongly enhanced resistance to M. grisea. Polymerase chain reaction (PCR) and Southern blot analysis confirmed that the T-DNA was integrated into the rice genome. T1 progenies generated from these plants were analysed for the presence of the hrf1 gene by PCR and inoculated with M. grisea for the segregation of resistance. The results showed that the resistance against M. grisea of the T1 progenies of the two lines segregated at a 3 : 1 ratio as a single Mendelian trait (Table 1). All the transgenic plants resistant to M. grisea were positive in PCR analysis for the presence of the hrf1 gene. The active harpin protein was isolated from the leaves of the T2 transgenic line NJH12, and a single band, identical in size to its original bacterial protein, was detected using Western blot analysis, indicating that the transferred hrf1 gene was expressed at detectable levels (Figure 1a). Protein levels increased in leaves during the growing season, reaching a maximum by flowering (data not shown).
Table 1. Segregation of resistance against Magnaporthe grisea in T1 progenies of transgenic rice lines NJH12 and NJH16*
A score of 0–9 was given based on the percentages of leaf area or branches that were necrotic as a result of infection by M. griseafor leaf blast and panicle blast, respectively. 0–4, resistant plants; 5–9, susceptible plants (see Table S4 for details).
3 : 1
3 : 1
hrf1 expression induces host defence responses
To determine whether hrf1 expression induces host defence responses similar to those produced by exogenous applications, the expression of eight defence-related genes was examined in four transgenic resistant T1 lines using reverse transcription (RT)-PCR. The genes tested were involved in plant basal defence pathways mediated by SA and JA (Dangl and Jones, 2001), and included six for defence (CHS, GLU, PR1a, PR1b, Chia4a and PAL) (Durrant and Dong, 2004) and two for signal transduction (COI1 and NPR1) (Table S1, see ‘Supplementary material’) (Spoel et al., 2003). The expression of OsPR1a, OsPR1b, Chia4a, PAL and NPR1 was constitutively enhanced in four T1 transgenic lines, but not in untransformed parental R109 plants (Figure 1b).
hrf1-transformed plants and their progenies confer nonspecific resistance to M. grisea in both controlled environments and the field
To determine whether hrf1 expression leads to resistance to M. grisea, untransformed R109 and all 29 transformed T1 lines were planted in a disease evaluation nursery at the National Centre for Resistant Crop Identification (NCRCI) in Pujiang County, Sichuan Province, China (Figure 2; Table S2, see ‘Supplementary material’). In addition to the diverse population of local isolates, dominated by isolates of the ZB race (National Coordinating Research Team on Rice Blast, 1980; Jin and Chai, 1990), a mixture of M. grisea races was also introduced at the seedling stage through the introduction of M. grisea-infested disease-inducing plants (Figure S2, see ‘Supplementary material’). All R109 plants exhibited severe leaf and panicle blast [disease severity (DS) = 9], and nine of the 29 transgenic lines, including NJH5, NJH12, NJH13 and NJH16, showed resistance to M. grisea (Figure 3a).
To examine whether hrf1 expression induces general resistance to different M. grisea races, seedlings of NJH12 (T2) and R109 were separately inoculated with six M. grisea isolates (ZB13, ZC3, ZD1, ZE3, ZF1 and ZG1) (Table S3, see ‘Supplementary material’). Humidity and temperature conditions were manipulated (relative humidity, > 95%; temperature, 26–28 °C) to optimize germination and appressorium formation of M. grisea. The isolates of ZB13, ZC3, ZD1 and ZG1 were the most virulent of the four major M. grisea races in rice-growing regions along the Yangtze River, whereas ZE3 and ZF1 were the most virulent isolates from the two dominant races (ZE and ZF) in northern China (Table S3) (National Coordinating Research Team on Rice Blast, 1980; Jin and Chai, 1990). Although R109 plants were highly susceptible to all six isolates (leaf blast; DS = 9.0), NJH12 plants were highly resistant to ZC3, ZD1 and ZG1, with no disease lesions being observed (Figure 3b). NJH12 plants were also resistant to ZB13 (DS = 2.7), but susceptible to ZE3 and ZF1 (DS = 9.0).
NJH12 (T3 and T5) progenies and untransformed parental R109 plants were further evaluated for their resistance to M. grisea in the field at NCRCI (Pujiang County, Sichuan Province, China) and at the National Centre for Rice Blast Resistance Identification (NCRBRI) in Anhou County, Hunan Province, China in 2003 and 2004, and T7 progenies of NJH12 plants were examined in Qianshan County, Anhui Province, China in 2005 (Figure 2). Leaf blast was very severe in Anhou County in 2003 (DS = 9.0), leading to the death of most R109 plants (> 95%) before flowering. Remarkably, T3 progenies of the NJH12 line grew well and matured normally in spite of high disease pressure (Figure 3c). Disease incidence, in general, was less severe in Pujiang County, but was not statistically evaluated because all plants were destroyed by a flood. In 2004, leaf blast was severe on plants of both R109 (DS = 8.0) and NJH12 (DS = 7.0) in Anhou County. However, although R109 plants exhibited extensive panicle blast (DS = 9.0), the T5 progenies of the NJH12 line remained healthy and matured normally with little evidence of panicle blast (Figure 4). In Pujiang County, leaf and panicle blast was very severe on R109 plants (DS = 9); however, NJH12 progenies were resistant to M. grisea and no panicle blast was observed (Figure 4). In 2005, severe rice blast killed most R109 plants, but T7 transgenic plants matured normally (Figure 3d). The consistently less severe disease incidence on panicles than on leaves suggests that hrf1-induced resistance increases as the plants mature, which is probably related to the level of harpin protein accumulation. Together, the results from both the specificity tests and field experiments directly illustrate that hrf1 expression in rice confers nonspecific resistance to all major M. grisea races in the Yangtze River region (Figure 2).
hrf1 expression alters M. grisea conidial germination and subsequent development in transgenic plants
Magnaporthe grisea has evolved a highly specialized structure, known as an appressorium, that is essential for foliar infection (Chumley and Valent, 1990; Dean, 1997; Talbot, 2003). M. grisea conidial germination and subsequent development were examined in transgenic and untransformed control plants. Leaves of NJH12 (T3) and R109 plants were inoculated with the conidia of the ZC3 race and monitored using a microscope for a period of 48 h (Liu and Dean, 1997). Marked differences in conidial development were consistently observed between NJH12 and the control R109. On R109 leaves, germ tube tips became enlarged by 24 h and heavily melanized appressoria were visible by 36 h (Figure 5). By contrast, conidia germinated abnormally on NJH12 leaves: multiple germ tubes formed by 30 h, no appressoria were visible by 36 h, and the conidiospores collapsed shortly thereafter (Figure 5). The inhibition of appressorium formation on NJH12 leaves appeared to occur prior to pathogen penetration.
hrf1 expression increases leaf silicon content in transgenic plants
Silicon contents were examined to determine whether hrf1 expression influenced leaf silicon concentrations, which often correlate with resistance to M. grisea (Rodrigues et al., 2003). At the tillering stage, the leaf silicon concentration was 69% higher in transgenic T7 progenies (9.54 ± 0.01%) than in untransformed R109 plants (5.62 ± 0.06%). At the final harvest, the silicon concentration in flag leaves was again significantly higher in NJH12 plants (18.8 ± 0.53%) than in the control (12.5 ± 0.51%).
Our results document consistent resistance in rice lines expressing the harpin gene hrf1 to diverse M. grisea races across large geographical distances in the most important rice-growing region in the world. Enhanced specific resistance to M. grisea has been reported recently in transgenic rice transformed with genes encoding antifungal compounds of both plant (Krishnamurthy et al., 2001) and microbial (Coca et al., 2004) origins. However, nonspecific resistance remains elusive, and no durable resistance in the field has so far been achieved in transgenic rice. To our knowledge, the results presented here are the first to report the nonspecific resistance of transgenic rice to M. grisea that is effective in several highly disease-conducive environments.
The high level of resistance in NJH12 plants to leaf and/or panicle blast and the inhibition of appressorium formation indicate that hrf1 expression may induce multiple resistance mechanisms. Enhanced expression of defence-related genes, which encode basic or acidic PR proteins, may be essential for hrf1-induced resistance to M. grisea in transgenic plants (Figure 1b). The NPR1 protein modulates defence pathways mediated by SA and JA (Spoel et al., 2003), and both induce SAR in rice against infection from blast fungus (Yang et al., 2004). In addition, the rice genes OsPR1a and OsPR1b are highly responsive to the rice blast pathogen (Agrawal et al., 2001), and constitutive expression of chitinase genes enhances blast resistance in rice (Nishizawa et al., 1999). Moreover, hrf1 expression in tobacco enhances resistance in T2 plants to Alternaria alternata and tobacco mosaic virus (Peng et al., 2004). We have recently transformed hrf1 into a major wheat cultivar (Yangmei 158) in the Yangtze River area, and have significantly enhanced the resistance in T1 plants to Fusarium head blight caused by Fusarium graminearium (data not shown). Together, these results suggest that hrf1-induced resistance involves general defence mechanisms that may function similarly across different plant species.
The inhibition of appressorium formation on NJH12 leaves appears to occur prior to pathogen penetration, suggesting that host plant defences are activated prior to infection. Higher silicon concentrations in transgenic plants may contribute to this inhibition, because silicon-mediated resistance to M. grisea in rice has been shown to correlate with a specific leaf cell reaction that interferes with the development of the fungus (Rodrigues et al., 2003). However, whether (and how) the enhanced expression of defence-related genes is related to the observed increase in leaf silicon concentration and the inhibition of appressorium formation remains to be investigated. Because harpinXoo itself does not influence the in vitro growth of M. grisea (data not shown), the inhibition of appressorium formation suggests that certain unidentified compounds produced by the transgenic plants may interfere with development in this fungus (Rodrigues et al., 2003).
In conclusion, our results illustrate that hrf1 expression confers nonspecific resistance in rice to M. grisea, probably through SAR and the activation of general defence responses that subsequently interfere with the growth and development of the pathogen. Over the last decade, numerous hrp genes have been identified in several Gram-negative phytopathogenic bacteria (Lee et al., 2001; Noel et al., 2002). It remains to be tested whether the expression of these genes will confer durable resistance to M. grisea and other pathogens of economic significance in rice or other crops, but the success of NJH12 transgenic rice reveals a clear pathway for the direction of further experimentation.
Further details of supporting information (Tables S1–S4; Figures S1 and S2) are given in ‘Supplementary material’.
hrf1 transformation and expression in rice
The full-length hrf1 gene was excised from pET30(a) with the restriction enzymes XbaI and BamHI and inserted into the corresponding sites in pBI121 between the cauliflower mosaic virus (CaMV) 35S promoter and the gusA gene (Sambrook et al., 1989). The resulting pBMH9 (or pBI-hrf1) (Figure S1) was sequenced to verify the correct orientation of the genes and transferred into Agrobacterium tumefaciens EHA105 using a freeze–thaw method (Huang et al., 2001). Rice (3-week-old seed calli) was transformed with pBMH9 by soaking the calli with A. tumefaciens suspensions, and co-cultivated for 2 days in N6 medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) and acetosyringone. Calli were then plated on N6 medium with 2,4-D, carbenicillin and kanamycin for selection. Resistant calli were transferred to Murashige and Skoog (MS) medium, and plantlets were then transferred to half-strength MS medium without hormones to induce root formation (Huang et al., 2001).
PCR analysis was performed using hrf1-specific primers (Zhu W.G. et al., 2000; Wen et al., 2003) and CaMV 35S promoter primers through the amplification of a 420-bp band of hrf1 and 800-bp band of the 35S promoter (Table S1). Expression of hrf1 in transgenic lines was examined using the protein extracts of leaves, and the concentration of proteins was estimated using the Bio-Rad reagent (Bio-Rad, Hercules, CA, USA). Western analysis was performed with anti-HRF1 antibodies (dual spiral), and total proteins were separated by 12% sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes. Membranes were incubated with anti-HRF1 antiserum and then with goat anti-rabbit alkaline phosphatase-conjugated immunoglobulin G (IgG). Protein bands were visualized using a Western blotting detection kit (Amersham, Buckinghamshire, UK) following the manufacturer's protocol.
RT-PCR evaluation of defence genes
Total RNA was prepared from well-expanded 6-week-old leaves using a Tripure kit (Roche, Indianapolis, IN, USA), following the manufacturer's instructions, and treated with RNase-free DNase (Promega, Madison, WI, USA). The EF1a gene was used as a standard and amplified with specific primers, resulting in a 495-bp product.
Plant materials and growth conditions
Rooting transgenic plants (T0) were transferred to soil in a growth chamber (12-h photoperiod; 28 °C; light strength, 30 000 lx), and a slow-release fertilizer was applied. Plants of each generation were self-pollinated to produce the subsequent generation. Seeds collected from T0 plants (i.e. T1) were grown in the field to maturity in Pujiang County, Sichuan Province, China. Genetic analysis of the segregation of resistance to M. grisea was conducted using T1 plants from NJH12 and NJH16. NJH12 progenies (T2, T4 and T6) and untransformed R109 plants were planted in Sanya, Hainan Province, China in the winter for generation advancement. T3 and T5 progenies were grown in Anhou County (Hunan Province, China) and Pujiang County (Sichuan Province, China), and T7 progenies were grown in Qianshan County (Anhui Province, China), during the regular rice-growing season (Table S2; Figure 2).
Specificity of NJH12 resistance to M. grisea races
The specificity of transgenic plant resistance to M. grisea races was determined at the Jiangsu Academy of Agricultural Sciences (JAAS), Nanjing, China. Resistant T1 plants were self-pollinated to obtain T2 generation plants, and a number of the T2 plants were used to determine the stability and specificity of the resistance to M. grisea races. M. grisea conidia were produced on a rice stalk powder agar medium (25 g rice stalk powder, 40 g cornmeal and 18 g agar per litre, pH 7.0), grown under fluorescent light and then in the dark to induce conidiation, and then harvested with washing. Three-week-old NJH12 (T2) and R109 plants were inoculated with M. grisea by spraying a fungal spore suspension (2 × 105 conidia/mL with 500 µg/mL Tween 20), maintained in the dark for 24 h and transferred to a controlled glasshouse. DS was recorded 7–10 days after M. grisea isolates (ZB13, ZC3, ZD1, ZE3, ZF1 and ZG1) were inoculated. The experiment was repeated twice, with 180 transgenic plants being examined each time (i.e. 30 plants for each M. grisea isolate).
Field evaluation of M. grisea resistance in transgenic plants and their progenies
The resistance of NJH12 progenies to M. grisea was examined in the field at NCRCI, Pujiang County, Sichuan Province, China (T1, T3 and T5) and NCRBRI, Anhou County, Hunan Province, China (T3 and T5) in 2003 and 2004. Surrounded by mountains, both sites experience prolonged foggy mornings and high diurnal temperature amplitudes, leading to local environmental conditions that highly favour M. grisea growth and infection. Isolates of ZB and ZC races of M. grisea dominate the natural population in Anhou County, and isolates of the ZB race are dominant in Pujiang County, although isolates of ZG and other races exist at both sites (National Coordinating Research Team on Rice Blast, 1980; Jin and Chai, 1990). The actual composition of M. grisea at each site was very diverse because rice cultivars, and presumably their associated pathogens, have been introduced across China over the last two decades. In addition to the diverse population of local isolates, a mixture of M. grisea races was also introduced at the seedling stage by way of M. grisea-infested, disease-inducing plants (Figure S2). In 2005, T7 progenies were further evaluated in the field in Qianshan County, Anhui Province, China (300 km west of Nanjing), where ZG isolates dominate. Because rice blast has been chronically severe in the last 15 years, disease-inducing plants were not introduced at this site.
For T3 and T5 progenies, NJH12 plots alternated with R109 plots in the field. To ensure a high density of M. grisea, experimental plots were encircled by disease-inducing plants (Figure S2). Disease-inducing plants were pre-infested with M. grisea and transplanted with test plants. All 50 plants in each plot were examined for leaf and panicle blast using very rigorous criteria, and each panicle was visually examined to estimate the percentages of branches that were necrotic as a result of infection by M. grisea, and given a rating from zero to nine (Table S4, see ‘Supplementary material’).
Microscopic examination of growth and infection of M. grisea pathogens on R109 and NJH12 plants
Freshly cut rice leaves (length, 5 cm) from 2–3-week-old R109 and NJH12 (T3) plants (one leaf for each plant) were taped at their tips to glass slides and placed face up on a layer of moistened paper in a clear culture box (Liu and Dean, 1997). Multiple 5-µL droplets of conidia (2 × 105 conidia/mL containing 500 µg/mL Tween 20) were placed on the leaf surface. Conidia, germ tubes and appressoria were stained in lactophenol-cotton blue solution. Appressorium formation was checked periodically. At each time period, 15 inoculation sites (five on each leaf) were examined microscopically for both transgenic and untransformed plants.
Leaf silicon concentration
Silicon concentrations in rice leaves were determined at the tillering stage and at final harvest. About 50 leaves from NJH12 (T7) and R109 plants were randomly harvested from each of the three field plots at the JAAS Experimental Station, Nanjing, China. Only fully grown leaves were collected at the tillering stage and flag leaves were sampled at the final harvest. The plants were grown on a clay soil with adequate silicon supply. Leaves were oven dried (70 °C for 7 days) and ground, and leaf silicon concentrations were determined and expressed as a percentage of the dry weight (Snyder, 2001).
We thank G.Y. Chen, X.B. Wang and H.S. Zhang for contributing to the design and execution of the field experiment. Thanks are also extended to F.H. Xiao and Y. Liu (Anhou County), D.H. Lu and G. Yao (Pujiang County) and T.C. Gao (Qianshan County) for evaluating M. grisea resistance, to Y.J. Zhou for providing the M. grisea conidia and access to the M. grisea evaluation facility at JAAS (Nanjing), and J.Y. Zhuang at the China National Rice Research Institute for silicon determination. We are also grateful to N.H. Cheng, G.A. Payne and H.D. Shew for their constructive comments, and to C. Tu for graph preparation. The project was supported by grants from the National Key Basic Research Programme of China (2003CB114204), the National Special Programme for Research and Industrialization of Transgenic Plants (JY03-B-12) and the National High Technology Research and Development Programme of China (2006AA10Z172).