Rice varieties with resistance to multiple races of Magnaporthe oryzae offer opportunities to manage rice blast in Australia

Rice blast caused by Magnaporthe oryzae is the most destructive disease of rice worldwide. Development of resistant varieties is considered as the most cost‐effective and sustainable way to manage rice blast. However, there remains a lack of knowledge about the resistance of rice varieties to blast disease in Australia. This study was conducted to determine if there was any resistance existing among the rice varieties grown in Australia to M. oryzae isolates from this country that belong to different races. There was a resistant reaction of the variety SHZ‐2 to all the five races of IA‐1, IA‐3, IA‐63, IB‐3 and IB‐59, with a percent disease index (%DI) less than 40. Varieties NTR587, BR‐IRGA‐409, Ceysvoni and Rikuto Norin 20 showed a resistant reaction to races IA‐3, IA‐63, IB‐3 and IB‐59; and the variety Kyeema exhibited a resistant reaction to races IA‐3, IB‐3 and IB‐59. For the races IA‐1 and IB‐59 with more than one isolate, varieties with differential disease reactions across different isolates belonging to the same race were also revealed: five varieties, Langi, Opus, Sherpa, Viet 1 and Topaz, exhibited differential disease reactions to the three IA‐1 isolates; 10 varieties showed differential disease reactions to the four IB‐59 isolates; in addition, the varieties that had differential disease reactions to the IA‐1 isolates also exhibited differential disease reactions to the IB‐59 isolates of race. This study provides valuable resistance sources for breeding programmes to develop rice varieties with resistance to multiple races of M. oryzae in Australia.     

Nitric oxide reduces Cu toxicity and Cu-induced NH4+ accumulation in rice leaves

Nitric oxide (NO) is a highly reactive, membrane-permeable free radical, which has recently emerged as an important antioxidant. Here we investigated the protective effect of NO against the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄ (10 mmol L⁻¹). It was found that free radical scavengers (sodium benzoate, thiourea, and reduced glutathione) reduced the toxicity and NH₄⁺ accumulation in rice leaves caused by excess CuSO₄. NO donor sodium nitroprusside (SNP) was also effective in reducing CuSO₄-induced toxicity and NH₄⁺ accumulation in rice leaves. The protective effect of SNP on the toxicity and NH₄⁺ accumulation can be reversed by 2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide, a NO scavenger, suggesting that the protective effect of SNP is attributable to NO released. Results obtained in the present study suggest that reduction of CuSO₄-induced toxicity and NH₄⁺ accumulation by SNP is most likely mediated through its ability to scavenge active oxygen species.     

Biochemical defence responses of resistant and susceptible rice genotypes against blast pathogen Magnaporthe oryzae

Abstract

Rice blast is the leading fungal disease which is caused by Magnaporthe oryzae that contributes for the significant decline in the rice yield throughout the globe. There is a need for the understanding of biochemical changes in rice plant during blast infection for the development of novel disease control strategies. In the present study, we isolated M. oryzae from the local paddy fields and the fungal isolates (VCF and PON) were identified by ITS-PCR using genomic DNA samples. Further, we inoculated resistant (BR2655 and TUNGA) and susceptible (INTAN and HR12) rice cultivars with PON and VCF isolates. PON isolate showed relatively high virulence compared to VCF and standard MTCC fungal strains. Therefore, we evaluated the effect of PON on the total protein content and plant defence-related key enzymes (peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, β-glucosidase, chitinase and lipoxygenase) activities between 24- and 120-hour post-inoculation (hpi). The results demonstrated the decrease in total protein content in all the inoculated cultivars. In addition, we observed the variation in the activity of peroxidase, polyphenol oxidase, β-glucosidase, chitinase and lipoxygenase at different time points in all the tested rice plants compared to respective controls. However, no significant difference was observed in the phenylalanine ammonia lyase activity relative to its control. Taken together, this study emphasizes on the variation in the activities of plant defence enzymes in different plant cultivars against the tested fungal pathogen and also implementation of defence enzymes as biochemical markers for resistant breeding.     

Tenuazonic acid,toxin of rice blast fungus,induces disease resistance and reactive oxygen production in plants

Effects of tenuazonic acid (TA) on rice leaf segments and on their interaction with compatible races of the blast fungus (Magnaporthe grisea, former name is Pyricularia oryzae) were studied. TA induced small brown necrotic spots on leaves Application of TA (1 or 5 mM) to leaves in mixtures with M. grisea spores induced a local disease resistance, which reduced the frequency of compatible lesions. TA was not fungitoxic but, in contact with the leaf, increased the capability of leaf diffusates to inhibit germination of M. grisea spores. In the infected leaves, the diffusate fungitoxicity was higher than in the healthy ones. Antioxidant enzymes, superoxide dismutase and catalase, and scavengers of hydroxyl radical, mannitol and formate, strongly inhibited the TA-induced diffusate fungitoxicity. It is suggested that the disease resistance induced by TA is mediated, at least partially, by generation of reactive oxygen species by rice leaves, which inhibit the development of the fungus directly or indirectly.     

The DnaJ protein OsDjA6 negatively regulates rice innate immunity to the blast fungus Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae (synonym: Pyricularia oryzae), severely reduces rice production and grain quality. The molecular mechanism of rice resistance to M. oryzae is not fully understood. In this study, we identified a chaperone DnaJ protein, OsDjA6, which is involved in basal resistance to M. oryzae in rice. The OsDjA6 protein is distributed in the entire rice cell. The expression of OsDjA6 is significantly induced in rice after infection with a compatible isolate. Silencing of OsDjA6 in transgenic rice enhances resistance to M. oryzae and also results in an increased burst of reactive oxygen species after flg22 and chitin treatments. In addition, the expression levels of WRKY45, NPR1 and PR5 are increased in OsDjA6 RNAi plants, indicating that OsDjA6 may mediate resistance by affecting the salicylic acid pathway. Finally, we found that OsDjA6 interacts directly with the E3 ligase OsZFP1 in vitro and in vivo. These results suggest that the DnaJ protein OsDjA6 negatively regulates rice innate immunity, probably via the ubiquitination proteasome degradation pathway.     

A Pid3 allele from rice cultivar Gumei2 confers resistance to Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases. Using map-based strategy and in silico approach we isolated a new rice (Oryza sativa L.) blast resistance allele of Pid3, designated Pi25, from a stable blast resistance cultivar Gumei2. Overexpression analysis and complementation test showed that Pi25 conferred blast resistance to M. oryzae isolate js001-20. Sequence analysis showed that Pi25 was an intronless gene of 2772 nucleotides with single nucleotide substitution in comparison to Pid3 at the nucleotide position 459 and predicatively encoded a typical coiled coil-nucleotide binding site-leucine rich repeat (CC--NBS--LRR) protein of 924 amino acid residuals with 100% identity to Pid3 putative protein. The susceptible allele pi25 in Nipponbare contained a nonsense mutation at the nucleotide position 2209 resulting in a truncated protein with 736 amino acid residuals. In addition, 14 nucleotide substitutions resulting in 10 amino acid substitutions were identified between Pi25 and pi25 upstream the premature stop codon in the susceptible allele. Although the mechanism of Pi25/Pid3-mediated resistance needs to be further investigated, the isolation of the allele would facilitate the utilization of Pi25/Pid3 in rice blast resistance breeding program via transgenic approach and marker assisted selection.     

Optimization of the HyPer sensor for robust real‐time detection of hydrogen peroxide in the rice blast fungus

Reactive oxygen species (ROS) production and breakdown have been studied in detail in plant‐pathogenic fungi, including the rice blast fungus, Magnaporthe oryzae; however, the examination of the dynamic process of ROS production in real time has proven to be challenging. We resynthesized an existing ROS sensor, called HyPer, to exhibit optimized codon bias for fungi, specifically Neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct could detect changes in fungal ROS during the plant infection process. Using confocal microscopy, we were able to visualize fluctuating ROS levels during the formation of an appressorium on an artificial hydrophobic surface, as well as during infection on host leaves. Using the plate reader, we were able to ascertain measurements of hydrogen peroxide (H₂O₂) levels in conidia as detected by the MoHyPer sensor. Overall, by the optimization of codon usage for N. crassa and related fungal genomes, the MoHyPer sensor can be used as a robust, dynamic and powerful tool to both monitor and quantify H₂O₂ dynamics in real time during important stages of the plant infection process.     

Twinfilin regulates actin assembly and Hexagonal peroxisome 1 (Hex1) localization in the pathogenesis of rice blast fungus Magnaporthe oryzae

Actin assembly at the hyphal tip is key for polar growth and pathogenesis of the rice blast fungus Magnaporthe oryzae. The mechanism of its precise assemblies and biological functions is not understood. Here, we characterized the role of M. oryzae Twinfilin (MoTwf) in M. oryzae infection through organizing the actin cables that connect to Spitzenkörper (Spk) at the hyphal tip. MoTwf could bind and bundle the actin filaments. It formed a complex with Myosin2 (MoMyo2) and the Woronin body protein Hexagonal peroxisome 1 (MoHex1). Enrichment of MoMyo2 and MoHex1 in the hyphal apical region was disrupted in a ΔMotwf loss-of-function mutant, which also showed a decrease in the number and width of actin cables. These findings indicate that MoTwf participates in the virulence of M. oryzae by organizing Spk-connected actin filaments and regulating MoHex1 distribution at the hyphal tip.     

Pex14/17, a filamentous fungus‐specific peroxin,is required for the import of peroxisomal matrix proteins and full virulence of Magnaporthe oryzae

Peroxisomes are ubiquitous organelles in eukaryotic cells that fulfil a variety of biochemical functions. The biogenesis of peroxisomes requires a variety of proteins, named peroxins, which are encoded by PEX genes. Pex14/17 is a putative recently identified peroxin, specifically present in filamentous fungal species. Its function in peroxisomal biogenesis is still obscure and its roles in fungal pathogenicity have not yet been documented. Here, we demonstrate the contributions of Pex14/17 in the rice blast fungus Magnaporthe oryzae (Mopex14/17) to peroxisomal biogenesis and fungal pathogenicity by targeting gene replacement strategies. Mopex14/17 has properties of both Pex14 and Pex17 with regard to its protein sequence. Mopex14/17 is distributed at the peroxisomal membrane and is essential for efficient peroxisomal targeting of proteins containing peroxisomal targeting signal 1. MoPEX19 deletion leads to the cytoplasmic distribution of Mopex14/17, indicating that the peroxisomal import of Pex14/17 is dependent on Pex19. The knockout mutants of MoPEX14/17 show reduced fatty acid utilization, reactive oxygen species (ROS) degradation and cell wall integrity. Moreover, Δmopex14/17 mutants show delayed conidial generation and appressorial formation, and a reduction in appressorial turgor accumulation and penetration ability in host plants. These defects result in a significant reduction in the virulence of the mutant. These data indicate that MoPEX14/17 plays a crucial role in peroxisome biogenesis and contributes to fungal development and pathogenicity.     

Effect of azoxystrobin and kresoxim‐methyl on rice blast and rice grain yield in China

Sensitivity to azoxystrobin and kresoxim‐methyl of 80 single‐spore isolates of Magnaporthe oryzae was determined. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting mycelial growth of the 80 M. oryzae isolates were 0.006–0.056 and 0.024–0.287 µg mL^?1, respectively. The EC₅₀ values for azoxystrobin and kresoxim‐methyl in inhibiting conidial germination of the M. oryzae populations were 0.004–0.051 and 0.012–0.105 µg mL^?1, respectively. There was significant difference in sensitivity to azoxystrobin or kresoxim‐methyl between the tested isolates representing differential sensitivity to carbendazim (MBC) and kitazin P (IBP); however, there was no correlation between this difference in sensitivity to azoxystrobin or kresoxim‐methyl and sensitivity to MBC or IBP, indicating that there was no cross‐resistance between azoxystrobin or kresoxim‐methyl and MBC or IBP. In the protective and curative experiments, kresoxim‐methyl exhibited higher protective and curative activity than azoxystrobin when applied at 150 and 250 µg mL^?1 accordingly, while azoxystrobin exhibited stronger inhibitory activity against M. oryzae isolates than that of kresoxim‐methyl in the in vitro test. The results of field experiments also suggested that both azoxystrobin and kresoxim‐methyl at 187.5 g.a.i. ha^?1 gave over 73% control efficacy in both sites, exhibiting excellent activity against rice blast. Taken together, azoxystrobin and kresoxim‐methyl could be a good substitute for MBC or IBP for controlling rice blast in China, but should be carefully used as they were both at‐risk.     

Phosphate excess increases susceptibility to pathogen infection in rice

Phosphorus (P) is an essential nutrient for plant growth and productivity. Due to soil fixation, however, phosphorus availability in soil is rarely sufficient to sustain high crop yields. The overuse of fertilizers to circumvent the limited bioavailability of phosphate (Pi) has led to a scenario of excessive soil P in agricultural soils. Whereas adaptive responses to Pi deficiency have been deeply studied, less is known about how plants adapt to Pi excess and how Pi excess might affect disease resistance. We show that high Pi fertilization, and subsequent Pi accumulation, enhances susceptibility to infection by the fungal pathogen Magnaporthe oryzae in rice. This fungus is the causal agent of the blast disease, one of the most damaging diseases of cultivated rice worldwide. Equally, MIR399f overexpression causes an increase in Pi content in rice leaves, which results in enhanced susceptibility to M. oryzae. During pathogen infection, a weaker activation of defence-related genes occurs in rice plants over-accumulating Pi in leaves, which is in agreement with the phenotype of blast susceptibility observed in these plants. These data support that Pi, when in excess, compromises defence mechanisms in rice while demonstrating that miR399 functions as a negative regulator of rice immunity. The two signalling pathways, Pi signalling and defence signalling, must operate in a coordinated manner in controlling disease resistance. This information provides a basis to understand the molecular mechanisms involved in immunity in rice plants under high Pi fertilization, an aspect that should be considered in management of the rice blast disease.     

4个太湖流域粳稻地方品种抗稻瘟病性的遗传分析

以太湖流域粳稻地方品种薄稻、铁杆青、江南晚和缺儿糯等广谱、高抗稻瘟病为材料, 与高感稻瘟病品种苏御糯杂交, 获得杂交F1、F2 , 分别接种日本稻瘟病鉴别菌系北1和中国稻瘟病菌生理小种ZE3、ZG1, 根据P1、P2、F1和F2等不同世代植株的抗、感反应, 分析地方品种对不同稻瘟病菌生理小种(菌系)的抗性遗传机理。结果表明: 薄稻、铁杆青及缺儿糯对北1菌系的抗性均可能由一对显性基因控制, 江南晚对北1的抗性则可能由两对抑制基因互作控制; 铁杆青及缺儿糯对ZE3小种的抗性均可能由一对显性基因控制, 薄稻和江南晚对ZE3小种的抗性可能分别由两对显性基因和两对抑制基因互作控制; 铁杆青对ZG1小种的抗性可能是由一对显性主基因控制, 薄稻和江南晚对ZG1小种的抗性则可能由两对抑制基因互作控制。进一步将薄稻与12个日本稻瘟病菌鉴别品种杂交,用北1菌系接种不同组合的F1和F2 , 进行抗病基因的等位性测定。结果表明, 薄稻对北1菌系的抗性基因与12个鉴别品种所携带的已知抗稻瘟病基因是不等位, 将该基因暂定为Pi-bd1(t)。     

OsSERK1 regulates rice development but not immunity to Xanthomonas oryzae pv. oryzae or Magnaporthe oryzae

Somatic embryogenesis receptor kinase (SERK) proteins play pivotal roles in regulation of plant development and immunity. The rice genome contains two SERK genes, OsSerk1 and OsSerk2. We previously demonstrated that OsSerk2 is required for rice Xa21-mediated resistance to Xanthomonas oryzae pv. oryzae (Xoo) and for normal development. Here we report the molecular characterization of OsSerk1. Overexpression of OsSerk1 results in a semi-dwarf phenotype whereas silencing of OsSerk1 results in a reduced angle of the lamina joint. OsSerk1 is not required for rice resistance to Xoo or Magnaporthe oryzae. Overexpression of OsSerk1 in OsSerk2-silenced lines complements phenotypes associated with brassinosteroid (BR) signaling defects, but not the disease resistance phenotype mediated by Xa21. In yeast, OsSERK1 interacts with itself forming homodimers, and also interacts with the kinase domains of OsSERK2 and BRI1, respectively. OsSERK1 is a functional protein kinase capable of auto-phosphorylation in vitro. We conclude that, whereas OsSERK2 regulates both rice development and immunity, OsSERK1 functions in rice development but not immunity to Xoo and M. oryzae.     

Inheritance of dsRNAs in the rice blast fungus, Magnaporthe grisea

The 1.6 and 1.8 kbp dsRNAs have been found in the rice blast fungus, Magnaporthe grisea strain MG01. These dsRNA molecules are located in cytoplasm of the fungal cells and maintained stably during vegetative growth. Three crosses between dsRNA free and dsRNA containing strains including a parental cross, sib-mating and back cross were made to follow the inheritance of dsRNAs during sexual reproduction. Approximately 10% of ascospore progenies (11 out of 105) contained dsRNAs from all three crosses. These data indicate that dsRNAs of M. grisea are inherited at a low frequency and not in a Mendelian fashion.     

Glutamate synthase MoGlt1‐mediated glutamate homeostasis is important for autophagy,virulence and conidiation in the rice blast fungus

Glutamate homeostasis plays a vital role in central nitrogen metabolism and coordinates several key metabolic functions. However, its function in fungal pathogenesis and development has not been investigated in detail. In this study, we identified and characterized a glutamate synthase gene MoGLT1 in the rice blast fungus Magnaporthe oryzae that was important to glutamate homeostasis. MoGLT1 was constitutively expressed, but showed the highest expression level in appressoria. Deletion of MoGLT1 resulted in a significant reduction in conidiation and virulence. The ΔMoglt1 mutants were defective in appressorial penetration and the differentiation and spread of invasive hyphae in penetrated plant cells. The addition of exogenous glutamic acid partially rescued the defects of the ΔMoglt1 mutants in conidiation and plant infection. Assays for MoAtg8 expression and localization showed that the ΔMoglt1 mutants were defective in autophagy. The ΔMoglt1 mutants were delayed in the mobilization of glycogens and lipid bodies from conidia to developing appressoria. Taken together, our results show that glutamate synthase MoGlt1‐mediated glutamate homeostasis is important for pathogenesis and development in the rice blast fungus, possibly via the regulation of autophagy.     

Endogenous salicylic acid protects rice plants from oxidative damage caused by aging as well as biotic and abiotic stress

Salicylic acid (SA) is a key endogenous signal that mediates defense gene expression and disease resistance in many dicotyledonous species. In contrast to tobacco and Arabidopsis, which contain low basal levels of SA, rice has two orders of magnitude higher levels of SA and appears to be insensitive to exogenous SA treatment. To determine the role of SA in rice plants, we have generated SA-deficient transgenic rice by expressing the bacterial salicylate hydroxylase that degrades SA. Depletion of high levels of endogenous SA in transgenic rice does not measurably affect defense gene expression, but reduces the plant's capacity to detoxify reactive oxygen intermediates (ROI). SA-deficient transgenic rice contains elevated levels of superoxide and H2O2, and exhibits spontaneous lesion formation in an age- and light-dependent manner. Exogenous application of SA analog benzothiadiazole complements SA deficiency and suppresses ROI levels and lesion formation. Although an increase of conjugated catechol was detected in SA-deficient rice, catechol does not appear to significantly affect ROI levels based on the endogenous catechol data and exogenous catechol treatment. When infected with the blast fungus (Magnaporthe grisea), SA-deficient rice exhibits increased susceptibility to oxidative bursts elicited by avirulent isolates. Furthermore, SA-deficient rice is hyperresponsive to oxidative damage caused by paraquat treatment. Taken together, our results strongly suggest that SA plays an important role to modulate redox balance and protect rice plants from oxidative stress.     

Proteins interacting with mitochondrial ATP‐dependent Lon protease (MAP1) in Magnaporthe oryzae are involved in rice blast disease

The ATP‐dependent Lon protease is involved in many physiological processes. In bacteria, Lon regulates pathogenesis and, in yeast, Lon protects mitochondia from oxidative damage. However, little is known about Lon in fungal phytopathogens. MAP1, a homologue of Lon in Magnaporthe oryzae, was recently identified to be important for stress resistance and pathogenesis. Here, we focus on a novel pathogenic pathway mediated by MAP1. Based on an interaction system between rice and a tandem affinity purification (TAP)‐tagged MAP1 complementation strain, we identified 23 novel fungal proteins from infected leaves using a TAP approach with mass spectrometry, and confirmed that 14 of these proteins physically interact with MAP1 in vivo. Among these 14 proteins, 11 candidates, presumably localized to the mitochondria, were biochemically determined to be substrates of MAP1 hydrolysis. Deletion mutants were created and functionally analysed to further confirm the involvement of these proteins in pathogenesis. The results indicated that all mutants showed reduced conidiation and sensitivity to hydrogen peroxide. Appressorial formations were not affected, although conidia from certain mutants were morphologically altered. In addition, virulence was reduced in four mutants, enhanced (with lesions forming earlier) in two mutants and remained unchanged in one mutant. Together with the known virulence‐related proteins alternative oxidase and enoyl‐CoA hydratase, we propose that most of the Lon‐interacting proteins are involved in the pathogenic regulation pathway mediated by MAP1 in M. oryzae. Perturbation of this pathway may represent an effective approach for the inhibition of rice blast disease.     

Modern elite rice varieties of the 'Green Revolution' have retained a large introgression from wild rice around the Pi33 rice blast resistance locus

During the breeding process of cultivated crops, resistance genes to pests and diseases are commonly introgressed from wild species. The size of these introgressions is predicted by theoretical models but has rarely been measured in cultivated varieties. By combining resistance tests with isogenic strains, genotyping and sequencing of different rice accessions, it was shown that, in the elite rice variety IR64, the resistance conferring allele of the rice blast resistance gene Pi33 was introgressed from the wild rice Oryza rufipogon (accession IRGC101508). Further characterization of this introgression revealed a large introgression at this locus in IR64 and the related variety IR36. The introgressed fragment represents approximately half of the short arm of rice chromosome 8. This is the first report of a large introgression in a cultivated variety of rice. Such a large introgression is likely to have been maintained during backcrossing only if a selection pressure was exerted on this genomic region. The possible traits that were selected are discussed.     

Temperature modulation and the presence of C20 fatty acids in mono‐ and digalactosyldiacylglycerol of Euglena gracilis and Lepocinclis acus: A modern interpretation of euglenid galactolipids using positive‐ion electrospray ionization/mass spectrometry

Mono‐ and digalactosyldiacylglycerol (MGDG and DGDG, respectively) are important galactolipids that comprise photosynthetic membranes in almost all photosynthetic organisms. Intact forms of MGDG and DGDG of Euglena gracilis and Lepocinclis acus, two example euglenids with secondary plastids of green algal origin, were elucidated with fatty acid regiochemistry via positive‐ion electrospray ionization/mass spectrometry at two growth temperatures. At 20°C, E. gracilis and L. acus produced predominantly 18:3/16:4 (sn‐1/sn‐2) MGDG, whereas at 30°C this was supplanted by 18:2/16:2 MGDG. At both temperatures were also observed a variety of other MGDG and DGDG forms, including C₂₀ fatty acid‐containing forms not expected in a green algal‐derived plastid. In addition to providing structural details of MGDG and DGDG not available in past studies, these results suggest a previously unknown relationship between these two organisms and the red algae. This study also illustrates that temperature modulation of galactolipids occurs via modification of unsaturation of both the sn‐1 and sn‐2 fatty acids; this is fundamentally different from previously published studies from our laboratory on other algal classes.