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.     

Physiological and cytological mechanisms of silicon-induced resistance in rice against blast disease

Rice (Oryza sativa L.) blast disease caused by Magnaporthe grisea is one of the most destructive diseases in the rice-growing areas of the world. Silicon is an important nutritional element especially for rice. Two near-isogenic lines of rice with different resistance to blast disease, i.e. CO39 (susceptible) and C101LAC (Pi-1) (resistant), were selected to determine the effects of Si amendment on the severity and incidence of rice blast disease. The physiological and cytological mechanisms involved in the induced disease resistance by silicon were investigated. Exogenous Si application at a concentration of 2 mM reduced the disease index by 45% for CO39 and 56% for C101LAC (Pi-1). Si application alone did not change lignin content and the activities of defense-related enzymes including peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) in rice leaves of both isogenic lines. However, after inoculation with M. grisea, Si-treated rice plants significantly increased the activities of POD, PPO and PAL in leaves of both isogenic lines. Si and lignin content were also significantly increased in Si-treated inoculated seedlings. Environmental scanning electron microscope observations revealed that Si amendment resulted in higher Si deposit on dumbbell bodies in the rice leaves and silicon papilla accumulation on the guard cell of stoma. These results suggest that silicon-induced defense response and cell silicification of rice leaves altogether contribute to the silicon-induced rice resistance to blast disease.     

Antioxidative enzymes and the Russian wheat aphid (Diuraphis noxia) resistance response in wheat (Triticum aestivum)

A crucial function of antioxidative enzymes is to remove excess reactive oxygen species (ROS), which can be toxic to plant cells. The effect of Russian wheat aphid (RWA), Diuraphis noxia (Mordvilko), infestation on the activities of antioxidative enzymes was investigated in the resistant (cv. Tugela DN) and the near-isogenic susceptible (cv. Tugela) wheat (Triticum aestivum L.). RWA infestation significantly induced the activity of superoxide dismutase, glutathione reductase and ascorbate peroxidase to higher levels in the resistant than in susceptible plants. These findings suggest the involvement of antioxidative enzymes in the RWA-wheat resistance response, which was accompanied by an early oxidative burst. The results are consistent with the role of ROS in the resistance response and the control of their levels to minimise toxic effects.     

Molecular mapping of rice blast resistance gene <Emphasis Type="Italic">Pi-1</Emphasis>(<Emphasis Type="Italic">t</Emphasis>) in the elite <Emphasis Type="Italic">indica</Emphasis> variety Samba mahsuri

Samba mahsuri (BPT 5204) is a cultivar of the medium slender grain indica variety of Oryza sativa grown across India for its high yield and quality. However, this cultivar is susceptible to several diseases and pests including rice blast. The analysis of near isogenic lines indicated the presence of a resistance gene, Pi-1(t) in the donor cultivar C101LAC which is highly resistant to the rice blast fungus Magnaporthe grisea (M. grisea). C101LAC was crossed with susceptible indica rice cultivar (BPT 5204) to generate the mapping population. A mendelian segregation ratio of 3:1 for resistant to susceptible F₂ plants using bulk segregation analysis confirmed the presence of a major gene pi-1(t) by simple sequence repeats marker RM224 to the highly virulent blast isolate DRR 001.     

Feeding‐induced changes in defence enzymes and PR proteins and their implications in host resistance to Nilaparvata lugens

Six rice genotypes showing susceptible and resistant reactions to brown planthopper (BPH), Nilaparvata lugens were studied for feeding‐induced changes in defence enzymes and pathogenesis‐related (PR) proteins. The high resistant genotypes PTB 33, ADT 45 and ASD 7 and moderately resistant genotypes CO 43 and KAU 1661 recorded the greater expression of defence enzymes peroxidase, polyphenol oxidase, phenylalanine ammonia lyase, total phenol and β‐1,3 glucanase in response to N. lugens feeding at 1 day after infestation (DAI) compared with susceptible genotype TN1. The greater activity of chitinase was observed in resistant cultivars at 3 DAI and the activity was sustained for more than 1 week compared with susceptible TN1. In conclusion, the current study revealed that these defence enzymes and PR proteins might attribute to the resistance mechanisms in rice plants against BPH infestation.     

Activity profile of defence-related enzymes in rice genotypes (Oryza sativa L.) against rice blast (Magnaporthe oryzae)

The control and infected leaf samples of blast resistant and susceptible rice genotypes were evaluated for activities of defence-related enzymes viz., total phenol content, chitinase, phenylalanine ammonia lyase (PAL), β-glycosidase, antioxidative enzymes, superoxide dismutase, peroxidase and ascorbate peroxidase. The level of total phenol content and the activity profile of chitinase, PAL and β-glycosidase significantly increased in both blast-resistant and susceptible rice genotypes with comparatively higher level induction Tetep, NLR-20104 and Swarnadhan the blast-resistant genotypes. The antioxidative enzymes were comparatively higher in the leaf samples of blast-resistant genotypes recording highest increase in NLR-20104 and KJT-5. The constitutive levels of total phenols and activity of defence-related and antioxidative enzymes in the control leaf samples differed among the genotypes and were even higher in the two blast susceptible genotypes (EK-70 and Chimansal). However, the level of induction as evident from the activity profile differences between control and infected leaf samples suggests higher level of induction was more which is indicative of the induced defence response. The genotype recording maximum induction of defence-related and antioxidative enzymes activity could be useful criteria in screening for blast resistant genotype in rice.     

水稻对稻负泥虫取食的生理响应

以水稻负泥虫-水稻互作为研究对象,分析了水稻负泥虫Oulema oryzae Kuwayama取食过程中抗、感2个品种水稻植株内保护性酶POD、SOD、CAT和MDA活性的变化动态.结果表明:在水稻负泥虫取食为害的过程中,SOD活性持续增强,POD、CAT和MDA活性呈现出先升高后降低的趋势,而且抗、感水稻植株中4种酶...     

AVR1-CO39 is a predominant locus governing the broad avirulence of Magnaporthe oryzae 2539 on cultivated rice (Oryza sativa L.)

Magnaporthe oryzae 2539 was previously found to be avirulent to most rice cultivars and, therefore, was assumed to carry many avirulence (AVR) genes. However, only one AVR gene, AVR1-CO39, which corresponds to a resistance (R) gene Pi-CO39(t) in rice cv. CO39, has been found from 2539 thus far. In order to identify more AVR genes, we isolated 228 progeny strains from a cross between 2539 and Guy11, an M. oryzae strain with strong virulence on rice, and inoculated these strains onto 23 rice accessions (22 individual cultivars and a mixture of 14 cultivars) that are all resistant to 2539 but susceptible to Guy11. Unexpectedly, the experimental results indicated that the avirulence of 2539 on these rice cultivars appeared to be controlled only by the AVR1-CO39 locus. Consistent with this result, we further found that all except one of the rice cultivars were resistant to two transformed Guy11 strains carrying a 1.05-kb fragment containing the AVR1-CO39 gene from 2539. These results suggest that AVR1-CO39 is a predominant locus controlling the broad avirulence of 2539 on cultivated rice. Based on the results of this study and other previous studies, we infer that AVR1-CO39 is a species-wise rather than a cultivar-wise host-specific AVR locus of M. oryzae for rice.     

Cytology of infection,development and expression of resistance to Plasmodiophora brassicae in canola

The timing and expression of resistance to four isolates of Plasmodiophora brassicae, collected from research sites where pathotypes 2, 3, 5 and 6 (Williams' system) had been dominant when characterised in 2006, were assessed in four new commercial cultivars of canola (Brassica napus) with resistance to clubroot. Each of the resistant cultivars was highly resistant to all four of the isolates, and there was no difference in their response to infection. Root hair infection occurred at high levels, but pathogen development occurred more slowly than in a susceptible cultivar (control). Secondary infection and development in cortical cells was severely inhibited in each of the resistant cultivars; only a few bi‐nucleated plasmodia were observed at 12 days after inoculation (DAI), and plasmodia were rarely observed at 18 and 24 DAI. In contrast, development in the susceptible cultivar had progressed to resting spores by 24 DAI. A dense ring of accumulated reactive oxygen species (ROS) was observed in the endodermis, pericycle and vascular cambium of non‐inoculated controls and inoculated plants of the resistant cultivars. However, the ROS ring disappeared rapidly in infected plants of the susceptible cultivar. Plasmodia invaded the stele of susceptible roots by preferentially colonising the xylem parenchyma cells. Expansion and enlargement of lignified xylem cells was observed by 35 DAI. The absence of any specific points of ROS accumulation or lignification of epidermal or cortical cells in the resistant cultivars indicates that a hypersensitive response is not the main mechanism of resistance in these lines. The uniform response of these resistant cultivars to the four isolates of P. brassicae indicates that the resistance in each cultivar may be conditioned by a gene(s) from a single source that confers broad resistance, because most sources of resistance to P. brassicae are pathotype specific.     

Molecular breeding: marker-assisted selection combined with biolistic transformation for blast and bacterial blight resistance in Indica rice (cv. CO39)

Based on blast pathogen population dynamics and lineage exclusion assays, we found that the major blast resistance genes Pi-1 and Piz-5 confer resistance against most Magnaporthe grisea lineages. Near-isogenic rice lines C101LAC and C101A51 carrying these two major genes for blast resistance in the background of a most blast-susceptible genotype were used for developing the pyramids. The closely linked RFLP marker RZ536 and NBS-LRR r10 marker for Pi-1 and a PCR-based SAP marker RG64 for Piz-5 were used to identify the genes in the parents and in marker-assisted breeding of the pyramided populations. To achieve multiple resistance against blast and blight in this cultivar, these blast-resistant pyramids were transformed with the cloned bacterial blight resistance gene Xa21 known to confer resistance to all races of Xanthomonas oryzae pv. oryzae (Xoo). Bioassays with six independent transformants showed that transgenic CO39 plants were resistant to both pathogens, M. grisea and Xoo. We report here the stacking of three major genes (Pi-1 + Piz-5 + Xa21) into rice using two different approaches of molecular breeding: marker-assisted selection (MAS) and genetic transformation.     

Oxidative stress, protein carbonylation, proteolysis and antioxidative defense system as a model for depicting water deficit tolerance in Indica rice seedlings

Water deficit is an important constraint to rice (Oryza sativa L.) productivity. The present study was undertaken to investigate whether the level of oxidative stress, carbonylation of proteins, proteolysis and status of antioxidative defense could serve as a model to distinguish water deficit tolerant and sensitive rice cultivars. When 10-day-grown seedlings of two rice cultivars, Malviya-36 (drought-sensitive) and Brown Gora (drought-tolerant) were subjected to ?1.0 and ?2.1 MPa water deficit treatments for 24–72 h with polyethylene glycol 6000 in the medium, a greater decline in the growth of the seedlings and levels of leaf water potential, relative water content, Chl a, Chl b, carotenoids and greater increase in leaf water loss were observed in the sensitive cultivar than the tolerant. Under similar level of water deficit seedlings of sensitive cultivar showed higher level of superoxide anion generation, H₂O₂, lipid peroxidation and proteolysis in roots as well as shoots compared to the tolerant. Drought-tolerant cultivar had higher constitutive level of antioxidative enzymes superoxide dismutase and catalase and the activities of these two enzymes alongwith of guaiacol peroxidase showed greater increase in this cultivar under water deficit compared to the sensitive. A significant decline in the level of protein thiol and a higher increase in protein carbonyls content, also confirmed by protein gel blot analysis with an antibody against 2,4-dinitrophenylhydrazine was observed in the seedlings of drought sensitive cv. Malviya-36 compared to the tolerant cv. Brown Gora when subjected to similar level of water deficit. Seedlings of drought sensitive cultivar, under water deficit, showed higher proteolytic activity, higher number of in-gel activity stained proteolytic bands and higher expression of oxidized proteins in roots compared to the tolerant cultivar. Results suggest that poor capacity of antioxidative enzymes could be, at least partly, correlated with water deficit sensitivity of sensitive cultivar and that higher activity of antioxidative enzymes superoxide dismutase, catalase, guaiacol peroxidase, low proteolytic activity, lower level of protein carbonyls and protein thiolation could serve as a model to depict water deficit tolerance in Indica rice seedlings.     

Effect of salicylic and abscisic acid administered through detached tillers on antioxidant system in developing wheat grains under heat stress

The mechanism imparting thermotolerance by salicylic acid (SA) and abscisic acid (ABA) is still unresolved using either spraying technique or in vitro conditions. Alternative way of studying these effects under near in vivo conditions is through the use of liquid culturing technique. Effects of SA and ABA (100 μM) on antioxidative enzymes, antioxidants and lipid peroxidation were studied in detached tillers of three wheat (Triticum aestivum L.) cultivars PBW 343, C 306 (heat tolerant) and WH 542 (heat susceptible) cultured in a liquid medium. Ears were subjected to heat shock treatment (45°C for 2 h) and then maintained at 25°C for 5 days. Heat shock treatment resulted in increased peroxidase (POD) activity, while superoxide dismutase (SOD) and catalase (CAT) activities were reduced compared to control. The decrease in CAT activity was more significant in susceptible cultivar WH 542. Concomitantly, content of α-tocopherol and lipid peroxides increased in heat-treated wheat ears, whereas contents of total ascorbate level were reduced. Following treatment with SA and ABA, activities of all three antioxidative enzymes increased in correspondence with an increase in ascorbate and α-tocopherol content. Apparently, lipid peroxide content was reduced by SA in heat tolerant cultivars (PBW 343 and C 306) whereas in susceptible cultivar it was decreased by ABA. The up-regulation of the antioxidant system by SA and ABA possibly contributes to better tolerance against heat shock-induced oxidative damage in wheat grains.     

Response of antioxidative enzymes to plum pox virus in two apricot cultivars

Recent evidence has indicated that activated oxygen species (AOS) may function as molecular signals in the induction of defence genes. In the present work, the response of antioxidative enzymes to the plum pox virus (PPV) was examined in two apricot (Prunus armeniaca L.) cultivars, which behaved differently against PPV infection. In the inoculated resistant cultivar (Goldrich), a decrease in catalase (CAT) as well as an increase in total superoxide dismutase (SOD) and dehydroascorbate reductase (DHAR) activities were observed. Ascorbate peroxidase (APX), glutathione reductase (GR) and monodehydroascorbate reductase (MDHAR) did not change significantly in relation to non-inoculated (control) plants. In the susceptible cultivar (Real Fino), inoculation with PPV brought about a decrease in CAT, SOD and GR, whereas a rise in APX, MDHAR and DHAR activities was found in comparison to non-inoculated (control) plants. Apricot leaves contain only CuZn-SOD isozymes, which responded differently to PPV depending on the cultivar. Goldrich leaves contained 6 SODs and both SOD 1 and SOD 2 increased in the inoculated plants. In leaves from Real Fino, 5 SODs were detected and only SOD 5 was increased in inoculated plants. The different behaviour of SODs (H2O2-generating enzymes) and APX (an H2O2-remover enzyme) in both cultivars suggests an important role for H2O2 in the response to PPV of the resistant cultivar, in which no change in APX activity was observed. This result also points to further studies in order to determine if an alternative H2O2-scavenging mechanism takes place in the resistant apricot cultivar exposed to PPV. On the other hand, the ability of the inoculated resistant cultivar to induce SOD 1 and SOD 2 as well as the important increase of DHAR seems to suggest a relationship between these activities and resistance to PPV. This is the first report about the effect of PPV infection on the antioxidative enzymes of apricot plants. It opens the way for the further studies, which are necessary for a better understanding of the role of antioxidative processes in viral infection by PPV in apricot plants.     

Comparative study of genes expressed from rice fungus-resistant and susceptible lines during interactions with Magnaporthe oryzae

Rice blast disease caused by Magnaporthe oryzae is the most important fungal disease of rice. To understand the molecular basis of interaction between the fungus and rice, we constructed a cDNA library from a rice-resistant line inoculated with M. oryzae. One hundred and fifty-three cDNA clones were sequence analyzed, of which 129 exhibited significant nucleotide sequence homology to known genes, 21 were homologous to unknown genes, while three clones did not match to any database. However, these three unmatched clones showed sequence homology at protein level in the protein databases and one of them encoded a disease resistance-related protein kinase and was abundant in the EST collection. Northern analysis showed that this disease resistance-related protein kinase gene was induced by inoculation and only expressed in the rice-resistant, but not susceptible, lines. Southern analysis showed that this gene was present in a single copy in the rice genome and co-segregated with the M. oryzae resistance in the cross of the resistant and susceptible lines. This study illustrates that sequencing of ESTs from inoculated resistant plants can reveal genes responsive to pathogen infection, which could help understand plant defense mechanisms.     

Vascular defense responses in rice: peroxidase accumulation in xylem parenchyma cells and xylem wall thickening.

The rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae is a vascular pathogen that elicits a defensive response through interaction with metabolically active rice cells. In leaves of 12-day-old rice seedlings, the exposed pit membrane separating the xylem lumen from the associated parenchyma cells allows contact with bacterial cells. During resistant responses, the xylem secondary walls thicken within 48 h and the pit diameter decreases, effectively reducing the area of pit membrane exposed for access by bacteria. In susceptible interactions and mock-inoculated controls, the xylem walls do not thicken within 48 h. Xylem secondary wall thickening is developmental and, in untreated 65-day-old rice plants, the size of the pit also is reduced. Activity and accumulation of a secreted cationic peroxidase, PO-C1, were previously shown to increase in xylem vessel walls and lumen. Peptide-specific antibodies and immunogold-labeling were used to demonstrate that PO-C1 is produced in the xylem parenchyma and secreted to the xylem lumen and walls. The timing of the accumulation is consistent with vessel secondary wall thickening. The PO-C1 gene is distinct but shares a high level of similarity with previously cloned pathogen-induced peroxidases in rice. PO-C1 gene expression was induced as early as 12 h during resistant interactions and peaked between 18 and 24 h after inoculation. Expression during susceptible interactions was lower than that observed in resistant interactions and was undetectable after infiltration with water, after mechanical wounding, or in mature leaves. These data are consistent with a role for vessel secondary wall thickening and peroxidase PO-C1 accumulation in the defense response in rice to X. oryzae pv. oryzae.