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.     

The mixture of kresoxim‐methyl and boscalid,an excellent alternative controlling grey mould caused by Botrytis cinerea

Grey mould, caused by the fungus Botrytis cinerea, is one of the most destructive diseases in greenhouses for which serious fungicide resistance has developed. Between 2003 and 2005, 213 isolates of B. cinerea from two geographical regions were characterised for baseline sensitivity to kresoxim‐methyl. In the absence of salicylhydroxamic acid (SHAM), the mean 50% effective concentration (EC₅₀) values were 6.67 ± 0.61 (mean ± SD) and 0.37 ± 0.10 mg L^?1 during growth and germination, respectively. In the presence of 100 mg L^?1 SHAM, baseline sensitivities were distributed as unimodal curves with mean EC₅₀ values of 2.38 ± 0.21 and 0.28 ± 0.09 mg L^?1 for inhibiting growth and inhibiting germination, respectively. The mixture of kresoxim‐methyl and boscalid showed good control efficacy against strawberry grey mould disease. After the mixture was extensively used on strawberry for 2 years, 50 isolates were collected and determined for their sensitivity to kresoxim‐methyl and boscalid, respectively. The mean EC₅₀ of germination inhibition by boscalid was 0.39 ± 0.08 mg L^?1. The mean EC₅₀ of germination inhibition by kresoxim‐methyl was 0.26 ± 0.07 mg L^?1 in the presence of 100 mg L^?1 SHAM. Sensitivities of B. cinerea to both kresoxim‐methyl and boscalid did not show any significant decrease. These results suggest that their mixture is a satisfactory alternative candidate for management of grey mould disease in greenhouses.     

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.     

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.     

Neck blast disease influences grain yield and quality traits of aromatic rice

A critical investigation was conducted to find out the effect of neck blast disease on yield-contributing characters, and seed quality traits of aromatic rice in Bangladesh. Both healthy and neck-blast-infected panicles of three aromatic rice cultivars (high-yielding and local) were collected and investigated at Plant Pathology Division, Bangladesh Rice Research Institute (BRRI), Gazipur, Bangladesh. All of the tested varieties were highly susceptible to neck blast disease under natural conditions, though no leaf blast symptoms appear on leaves. Neck blast disease increased grain sterility percentages, reduced grain size, yield and quality traits of seeds. The degrees of yield and seed quality reduction depended on disease severity and variety's genetic make-up. Unfilled grains were the main source of seed-borne pathogen, especially for blast in the seed lot. Transmission of blast pathogen from neck (panicle base) to seed was very poor. These findings are important, especially concerning the seed certification programme in which seed lots are certified on the basis of field inspection. Finally, controlled experiments are needed to draw more critical conclusions.     

A genome‐wide survey reveals abundant rice blast R genes in resistant cultivars

Plant resistance genes (R genes) harbor tremendous allelic diversity, constituting a robust immune system effective against microbial pathogens. Nevertheless, few functional R genes have been identified for even the best‐studied pathosystems. Does this limited repertoire reflect specificity, with most R genes having been defeated by former pests, or do plants harbor a rich diversity of functional R genes, the composite behavior of which is yet to be characterized? Here, we survey 332 NBS‐LRR genes cloned from five resistant Oryza sativa (rice) cultivars for their ability to confer recognition of 12 rice blast isolates when transformed into susceptible cultivars. Our survey reveals that 48.5% of the 132 NBS‐LRR loci tested contain functional rice blast R genes, with most R genes deriving from multi‐copy clades containing especially diversified loci. Each R gene recognized, on average, 2.42 of the 12 isolates screened. The abundant R genes identified in resistant genomes provide extraordinary redundancy in the ability of host genotypes to recognize particular isolates. If the same is true for other pathogens, many extant NBS‐LRR genes retain functionality. Our success at identifying rice blast R genes also validates a highly efficient cloning and screening strategy.     

稻瘟菌无毒基因研究进展

稻瘟菌是引起水稻稻瘟病的病原物。水稻与稻瘟菌间存在广泛而特异的相互作用,是研究寄主与病原物互作的重要模式系统。本文对稻瘟菌与水稻互作最重要的激发子―无毒基因的研究现状进行了概括,讨论了无毒基因的定位、克隆方法以及已克隆无毒基因的功能及进化研究,同时对今后无毒基因研究的重要方向进行了探讨,为深入理解无毒基因的功能及与水稻可能的互作关系奠定了基础。     

Rmg8 and Rmg7, wheat genes for resistance to the wheat blast fungus,recognize the same avirulence gene AVR‐Rmg8

Rmg8 and Rmg7 are genes for resistance to the wheat blast fungus (Pyricularia oryzae), located on chromosome 2B in hexaploid wheat and chromosome 2A in tetraploid wheat, respectively. AVR‐Rmg8, an avirulence gene corresponding to Rmg8, was isolated from a wheat blast isolate through a map‐based strategy. The cloned fragment encoded a small protein containing a putative signal peptide. AVR‐Rmg8 was recognized not only by Rmg8, but also by Rmg7, suggesting that these two resistance genes are equivalent to a single gene from the viewpoint of resistance breeding.     

In vitro selection of resistant rice plants against rice blast caused by Pyricularia oryzae via tissue culture technique

Abstract

A step by step protocol for resistant calli selection via a tissue culture technique under stress of Pyricularia oryzae culture filtrates was followed. Rice embryos dissected apart from the endosperm of susceptible rice seeds (Giza 176 and Riho) to P. oryzae produced embryonic calli on media containing various growth regulators of 2,4-D at concentrations of 0, 1, 1.5 and 2 mg/L and/or benzyl amino purine (BAP) at 0, 0.5, 1 and 1.5 mg/L when incubated under complete dark conditions for three weeks. Embryonic explants only produced shoots on media containing BAP. Selection of resistant calli was carried out in vitro under the challenging stress of increasing concentration of the pathogen P. oryzae culture filtrate (CF) from “0” up to 100%. The selection protocol has two directions. The first is step-by-step selection from lower to higher selective (CF) concentrations. The second is the exchangeable continuous cycles with and without the same selective (CF) concentration until the end of the selection regime to avoid calli adaptation to (CF). The regenerated calli to plantlets occurred under (CF) stress showed resistance and susceptibility when exposed to the pathogen infection under greenhouse conditions. The results reveal that the resistance in regenerated rice plantlets to P. oryzae pathogen segregated as 1 resistant: 2 moderate resistant: 1 susceptible giving the predication that the resistance in rice to P. oryzae may be controlled by one pair of genes. The in vitro selective regime via tissue cultures is advisable for the selection of novel disease resistant plants because of its time saving, space, money, it is easily applied and has a bio-safe approach.     

Mitochondrial dynamics and mitophagy are necessary for proper invasive growth in rice blast

Magnaporthe oryzae causes blast disease, which is one of the most devastating infections in rice and several important cereal crops. Magnaporthe oryzae needs to coordinate gene regulation, morphological changes, nutrient acquisition and host evasion in order to invade and proliferate within the plant tissues. Thus far, the molecular mechanisms underlying the regulation of invasive growth in planta have remained largely unknown. We identified a precise filamentous-punctate-filamentous cycle in mitochondrial morphology during Magnaporthe–rice interaction. Interestingly, disruption of such mitochondrial dynamics by deletion of genes regulating either the mitochondrial fusion (MoFzo1) or fission (MoDnm1) machinery, or inhibition of mitochondrial fission using Mdivi-1 caused significant reduction in M. oryzae pathogenicity. Furthermore, exogenous carbon source(s) but not antioxidant treatment delayed such mitochondrial dynamics/transition during invasive growth. In contrast, carbon starvation induced the breakdown of the mitochondrial network and led to more punctate mitochondria in vitro. Such nutrient-based regulation of organellar dynamics preceded MoAtg24-mediated mitophagy, which was found to be essential for proper biotrophic development and invasive growth in planta. We propose that precise mitochondrial dynamics and mitophagy occur during the transition from biotrophy to necrotrophy and are required for proper induction and establishment of the blast disease in rice.     

Galactolipid depletion in blast fungus‐infected rice leaves

This research focuses on galactolipid depletion in blast fungus‐infected rice leaves. Two major galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), from rice leaves were isolated and purified. The chemical structure of MGDG was identified as 1,2‐dilinolenyl‐3‐O‐β‐d ‐galactopyranosyl‐sn‐glycerol, and that of DGDG as 1,2‐dilinolenyl‐3‐O‐[α‐d ‐galactopyranosyl‐(1→6)‐O‐β‐d ‐galactopyranosyl]‐sn‐glycerol. Both the MGDG and DGDG content in the incompatible blast fungus race‐infected leaves decreased more than those in the compatible blast fungus race‐infected leaves during the infection process. Active oxygen species had the ability to peroxygenate and de‐esterify MGDG or DGDG in vitro, suggesting that active oxygen species play an important role in galactolipid depletion during the process of rice blast fungus invasion. Other possible functions of rice galactolipids during disease resistance are also discussed.     

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.     

Enhancing blast disease resistance by overexpression of the calcium‐dependent protein kinase OsCPK4 in rice

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium‐dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.     

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.     

Comparative proteomic analysis of methyl jasmonate‐induced defense responses in different rice cultivars

Jasmonate is an important endogenous chemical signal that plays a role in modulation of plant defense responses. To understand its mechanisms in regulation of rice resistance against the fungal pathogen Magnaporthe oryzae, comparative phenotype and proteomic analyses were undertaken using two near‐isogenic cultivars with different levels of disease resistance. Methyl‐jasmonate (MeJA) treatment significantly enhanced the resistance against M. oryzae in both cultivars but the treated resistant cultivar maintained a higher level of resistance than the same treated susceptible cultivars. Proteomic analysis revealed 26 and 16 MeJA‐modulated proteins in resistant and susceptible cultivars, respectively, and both cultivars shared a common set of 13 proteins. Cumulatively, a total of 29 unique MeJA‐influenced proteins were identified with many of them known to be associated with plant defense response and ROS accumulation. Consistent with the findings of proteomic analysis, MeJA treatment increased ROS accumulation in both cultivars with the resistant cultivar showing higher levels of ROS production and cell membrane damage than the susceptible cultivar. Taken together, our data add a new insight into the mechanisms of overall MeJA‐induced rice defense response and provide a molecular basis of using MeJA to enhance fungal disease resistance in resistant and susceptible rice cultivars.     

Differential accumulation of γ‐aminobutyric acid in elicited cells of two rice cultivars showing contrasting sensitivity to the blast pathogen

Intracellular free amino acid pools were quantified in suspension cultured cells of a blast‐sensitive and a blast‐resistant rice genotype at increasing times after treatment with Magnaporthe oryzae cell wall hydrolysates. Besides some expected variations in free phenylalanine, a remarkable early increase of γ‐aminobutyric acid (GABA) levels was evident in both cultivars. Glutamate decarboxylase activity and protein levels were unaffected. GABA homeostasis was recovered in the sensitive cultivar 48 h after the treatment. In contrast, a further GABA accumulation and a general increase of most amino acids was found at this later stage in the resistant genotype, which showed a larger decrease in cell viability as a consequence of elicitor addition. Data support a recently hypothesised role of GABA metabolism in the plant response to fungal pathogens.