Proteomic analysis of bacterial-blight defense-responsive proteins in rice leaf blades

Plants exhibit resistance against incompatible pathogens, via localized and systemic responses as part of an integrated defense mechanism. To study the compatible and incompatible interactions between rice and bacteria, a proteomic approach was applied. Rice cv. Java 14 seedlings were inoculated with compatible (Xo7435) and incompatible (T7174) races of Xanthomonas oryzae pv. oryzae (Xoo). Cytosolic and membrane proteins were fractionated from the leaf blades and separated by 2-D PAGE. From 366 proteins analyzed, 20 were differentially expressed in response to bacterial inoculation. These proteins were categorized into classes related to energy (30%), metabolism (20%), and defense (20%). Among the 20 proteins, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (RuBisCO LSU) was fragmented into two smaller proteins by T7174 and Xo7435 inoculation. Treatment with jasmonic acid (JA), a signaling molecule in plant defense responses, changed the level of protein accumulation for 5 of the 20 proteins. Thaumatin-like protein and probenazole-inducible protein (PBZ) were commonly up-regulated by T7174 and Xo7435 inoculation and JA treatment. These results suggest that synthesis of the defense-related thaumatin-like protein and PBZ are stimulated by JA in the defense response pathway of rice against bacterial blight.     

A mutation in an exoglucanase of Xanthomonas oryzae pv. oryzae,which confers an endo mode of activity,affects bacterial virulence,but not the induction of immune responses,in rice

Xanthomonas oryzae pv. oryzae (Xoo) causes bacterial blight, a serious disease of rice. Xoo secretes a repertoire of cell wall‐degrading enzymes, including cellulases, xylanases and pectinases, to degrade various polysaccharide components of the rice cell wall. A secreted Xoo cellulase, CbsA, is not only a key virulence factor of Xoo, but is also a potent inducer of innate immune responses of rice. In this study, we solved the crystal structure of the catalytic domain of the CbsA protein to a resolution of 1.86 Å. The core structure of CbsA shows a central distorted TIM barrel made up of eight β strands with N‐ and C‐terminal loops enclosing the active site, which is a characteristic structural feature of an exoglucanase. The aspartic acid at the 131st position of CbsA was predicted to be important for catalysis and was therefore mutated to alanine to study its role in the catalysis and biological functions of CbsA. Intriguingly, the D131A CbsA mutant protein displayed the enzymatic activity of a typical endoglucanase. D131A CbsA was as proficient as wild‐type (Wt) CbsA in inducing rice immune responses, but was deficient in virulence‐promoting activity. This indicates that the specific exoglucanase activity of the Wt CbsA protein is required for this protein to promote the growth of Xoo in rice.     

The molecular basis of disease resistance in rice

The rice gene Xa21 conferring resistance to Xanthomonas oryzae pv. oryzae (Xoo), was isolated using a map-based cloning strategy. Compared with previously cloned genes, the structure of Xa21 represents a novel class of plant disease R genes encoding a putative receptor kinase (RK). This article proposes a model for the mode of action of Xa21 and summarizes our current knowledge of the modular basis of resistance in rice to bacterial leaf blight and blast.     

Two thiadiazole compounds promote rice defence against Xanthomonas oryzae pv. oryzae by suppressing the bacterium's production of extracellular polysaccharides

Thiazole, isothiazole, thiadiazole, and their derivatives are used to control various human, animal and plant diseases. In addition to having direct anti‐microbial and anti‐fungal properties, these compounds are thought to induce host defences, but the mechanism of defence induction remains poorly understood. This article reports that the thiadiazoles of zinc thiazole and bismerthiazol induce H₂O₂ accumulation, up‐regulation of defence‐related genes, callose deposition and hypersensitive response‐like cell death in rice leaves infected with Xanthomonas oryaze pv. oryzae (Xoo) strain ZJ173, but not in non‐infected leaves. These defence responses in Xoo‐infected leaves were suppressed by the exogenous application of catalase, which reduces H₂O₂ accumulation. The application of extracellular polysaccharides (EPSs) extracted from strain ZJ173 significantly compromised rice defence against ZJ173 with or without thiadiazole treatment. The EPS‐deficient Xoo mutant ?gumH triggered a stronger defence than its parent strain ZJ173. The thiadiazole treatments reduced EPS production by strain ZJ173, but not by the thiadiazole‐resistant strain 2‐1‐1, which is thiadiazole resistant in vivo, but not in vitro; moreover, enhanced defence was not detected in thiadiazole‐treated rice inoculated with 2‐1‐1. Based on these data, we infer that zinc thiazole and bismerthiazol promote rice defence against Xoo by inhibiting the production of bacterial EPS.     

Development of pea bacterial blight caused by Pseudomonas syringae pv. pisi in winter and spring cultivars of combining peas (Pisum sativum) with different sowing dates

Pea bacterial blight occurred by natural infection in a field trial on peas in 1995. Disease development in the winter cultivars Rafale, Frilene and Froidure was compared with that in the spring cultivars Baccara, Conquest and Bohatyr, each sown on six dates in October, November, December, mid-March, late March and April. Disease incidence had reached 100% plants affected in all treatments by mid-July. Disease severity was greater in winter-sown (October, November or December) than in spring-sown peas of each cultivar at each assessment. Significant (P < 0.05) differences in disease severity occurred between cultivars in the winter-sown plots in May and June and the spring cultivars were affected more severely than the winter cultivars. Comparison of areas under the disease progress curves for both disease incidence and severity also showed that the winter-sown peas were more affected by disease than spring-sown peas and that spring cultivars were more severely affected than winter cultivars. Yield was strongly correlated with disease severity. A linear regression model suggested that, for peas sown in October, November or December, a yield loss of 0.5 tha^-1 occurred for each 10% increase in canopy area affected by pea bacterial blight.     

Biological control of rice bacterial blight by Lysobacter antibioticus strain 13-1

Bacterial leaf blight (BB) is a worldwide destructive rice disease caused by pathogen Xanthomonas oryzae pv. oryzae (Xoo). A novel strain of Lysobacter antibioticus, which was isolated from the rhizosphere of rice in Yunnan Province of China, can significantly inhibit the growth of various phytopathogenic bacteria and fungi, especially BB pathogen Xoo. In greenhouse experiments, whole bacterial broth culture (WBC) of strain 13-1 was more effective in reducing BB than other components of the culture, with disease suppression efficiency up to 69.7%. However, bacterial cells re-suspended in water, cell-free culture extracts, and heated cultures also significantly reduced BB severity. Suppression efficiencies ranged from 79.0% to 61.8% for undiluted to 100-fold dilution treatments and from 57.6% to 31.7% when the WBC of strain 13-1 (10⁸ CFU/mL) was applied at 3 days and 7 days prior to pathogen inoculation, respectively. In three field trials, strain 13-1 reduced BB incidence by 73.5%, 78.3%, and 59.1%, respectively. Disease suppression by strain 13-1 varied significantly among different rice cultivars, although efficacy was not directly related to the susceptibility level of the cultivars. Efficacy of biocontrol was also affected by different pathogen isolates, with some isolates of Xoo being more sensitive to 13-1 suppression than others. These results suggest that antibiotics and density of colonization on leaves may be involved for biological control of rice BB by strain 13-1. To our knowledge, this is the first report of L. antibioticus being a potential biocontrol agent for rice bacterial blight.     

Identification of a YAC clone carrying the Xa-1 allele, a bacterial blight resistance gene in rice

Map-based cloning methods have been applied for isolation of Xa-1, one of the bacterial blight resistance genes in rice.Xa-1 was previously mapped on chromosome 4 using molecular markers. For positional cloning of Xa-1, a high-resolution genetic map was made for theXa-1 region using an F₂ population of 402 plants and additional molecular markers. Three restriction fragment length polymorphism (RFLP) markers, XNpb235, XNpb264 and C600 were found to be linked tightly to Xa-1, with no recombinants, and U08₇₅₀ was mapped 1.5 cM from Xa-1. The screening of a yeast artificial chromosome (YAC) library using theseXa-1-linked RFLP markers resulted in the identification of ten contiguous YAC clones. Among these, one YAC clone, designated Y5212, with an insert of 340 kb, hybridized with all three tightly linked markers. This YAC was confirmed to possess the Xa-1 allele by mapping the Xa-1 gene between both end clones of this YAC (Y5212R and Y5212L).     

Studies on a Quantitative Analysis of Oryzalides and Oryzalic Acids in Rice Plants by GC-SIM

A procedure for the quantitative analysis of oryzalides and oryzalic acids in leaves of rice cultivars was established by using GC-MS-selective ion monitoring (GC-SIM), 17-deuterated oryzalide B being used as an internal reference. The total contents of four compounds of oryzalides and oryzalic acids in the leaves at three different growth stages of Norin-27, i.e., seedling, tillering, and ripening, showed values of 5.3, 17.9, and 37.9 μg/g, respectively. The contents of oryzalides and oryzalic acids in each organ, i.e., leaf, stem, ear, and root, showed values of 35.0 and 1.7 μg/g for the former two, respectively, and were not detectable for the latter two. A preliminary analysis of Norin-27 and Rantai emas leaves inoculated with an incompatible and compatible strain of Xanthomonas campestris pv. oryzae showed a greater accumulation of oryzalides and oryzalic acids, especially in the lesion area, than the figures for healthy leaves. Mechanical injury by a needle-bundle also increased the total amounts.     

Identification and expression analysis of components involved in rice Xa21‐mediated disease resistance signalling

Rice Xa21 gene encodes a receptor‐like kinase that confers broad‐spectrum resistance against Xanthomonas oryzae pv. oryzae (Xoo). Recently, a number of genes involved in the Xa21‐mediated disease resistance pathway have been identified. Based on our previous data and the literature, we chose 16 candidate proteins and made corresponding antibodies. Using Western blotting, we systematically investigated the expression profile of the proteins in Xa21‐mediated disease resistance response. We found nine proteins with altered expression. We further compared their expression in resistance, susceptible and mock responses, and found that GST expression was up‐regulated during the resistance process, indicating GST is a positive regulator in resistance responses. ATPsB expression was down‐regulated during both the resistance and susceptible response processes, although it was higher in the resistance response than that in the susceptible response. The total amount of MYB, GAPDH, CatB, Trx and NB‐ARC proteins was lower in the resistance than in the susceptible response, but their abundance per unit bacteria in the resistance response was still higher than in the susceptible response, suggesting that these proteins might be positive regulators in the resistance response. In addition, expression of another ERF was induced by inoculation with bacterial blight pathogen, and expression of Zf‐LSD1 was activated by wounding stress alone. Interestingly, most proteins showed similar altered expression patterns in the resistance and susceptible responses, but differed to some extents, implying that both responses might share common molecular mechanisms. This study revealed evidence of resistance‐related protein expression, providing a foundation for better understanding of their functions.     

Loss of chloroplast‐localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection

Protein phosphatases (PPs) counteract kinases in reversible phosphorylation events during numerous signal transduction pathways in eukaryotes. PP2Cs, one of the four major classes of the serine/threonine‐specific PP family, are greatly expanded in plants. Thus, PP2Cs are thought to play a specific role in signal transduction pathways. Some rice PP2Cs classified in subgroup K are responsive to infection by the compatible Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight. In Arabidopsis thaliana, orthologous PP2C genes (AtPP2C62 and AtPP2C26) classified to subgroup K are also responsive to Xanthomonas campestris pv. campestris (Xcc, causal agent of black rot) infection. To elucidate the function of these subgroup K PP2Cs, atpp2c62‐ and atpp2c26‐deficient A. thaliana mutants were characterized. A double mutant plant which was inoculated with a compatible Xcc showed reduced lesion development, as well as the suppression of bacterial multiplication. AtPP2C62 and AtPP2C26 localized to the chloroplast. Furthermore, the photosynthesis‐related protein, chaperonin‐60, was indicated as the potential candidate for the dephosphorylated substrate catalysed by AtPP2C62 and AtPP2C26 using two‐dimensional isoelectric focusing sodium dodecylsulfate‐polyacrylamide gel electrophoresis (2D‐IDF‐SDS‐PAGE). Taken together, AtPP2C62 and AtPP2C26 are suggested to be involved in both photosynthesis and suppression of the plant immune system. These results imply the occurrence of crosstalk between photosynthesis and the plant defence system to control productivity under pathogen infection.     

B-type natriuretic peptide and anthropometric measures in a Brazilian elderly population with a high prevalence of Trypanosoma cruzi infection

B-type natriuretic peptide (BNP) is a diagnostic and prognostic tool in heart failure and also in Chagas disease, which is caused by the protozoan Trypanosoma cruzi and has cardiomyopathy as a main feature. BNP lipolytic actions and T. cruzi infection in the adipose tissue have been recently described. We aim to investigate the relationship between BNP and anthropometric measures and whether it is influenced by T. cruzi infection. We measured BNP, body mass index (BMI), waist circumference (WC), triceps skin-fold thickness (TSF) and performed serological, biochemical and electrocardiographic exams in 1398 subjects (37.5% infected with T. cruzi) in a community-dwelling elderly population in Bambui city, Brazil. Linear multivariate regression analysis was performed to investigate determinants of BNP levels. BNP levels were significantly (p < 0.05) higher in T. cruzi-infected subjects than in the non-infected group (median = 121 and 64 pg/mL, respectively). BMI, WC and TSF in infected subjects were significantly lower than those in non-infected subjects (24.3 vs. 25.5 kg/m2; 89.2 vs. 92.4 cm; and 14.5 vs. 16.0 mm, respectively). There was an inverse relationship between BNP levels and BMI (b = −0.018), WC (b = −0.005) and TSF (b = −0.193) levels. Infected and non-infected groups showed similar inverse relationships between BNP and BMI (b = −0.021 and b = −0.015, respectively). In conclusion, there was an inverse relationship between BNP levels and the anthropometric measures. Despite the actions in the adipose tissue, T. cruzi infection did not modify the associations between BNP and BMI, suggesting that body mass does not modify the accuracy of BNP in Chagas disease.     

Endophytic yeast diversity in leaf tissue of rice,corn and sugarcane cultivated in Thailand assessed by a culture-dependent approach

Endophytic yeasts are yeast that can colonize healthy plant tissues without causing any damage to the host plant. This work aimed to explore the diversity of endophytic yeasts in leaf tissue of main agricultural crops (rice, corn and sugarcane) in Thailand, by a culture-dependent approach. A total of 311 leaf samples, consisting of rice (n = 100), corn (n = 109) and sugarcane (n = 102). From the tissue of rice (n = 92), corn (n = 76) and sugarcane (n = 78) leaf samples, 117, 118 and 123 yeast strains were respectively isolated and identified based on the D1/D2 region of the large subunit (LSU) rRNA gene sequence analysis to be yeast species in both the phyla Basidiomycota and Ascomycota. Higher numbers of basidiomycetous yeast than ascomycetous yeast were detected in the leaf tissue of the three crops. Pseudozyma (Dirkmeia) churashimaensis (Ustilaginales) was the most prevalent yeast species in the rice and corn leaves with relative frequencies (RF) of 35.9 % and 17.8 %, respectively. Whereas the predominant species in the sugarcane leaves was Meyerozyma caribbica (Saccharomycetales) with an RF of 14.6 %. In addition, six new yeast species and one new yeast genus were proposed. Our findings suggest that these plant species are good sources from which new yeast species may be isolated.     

Suppression of mouse lymphocyte responses to mitogens in vitro by Trypanosoma cruzi

Maleckar J. R. and Kierszenbaum F. 1984. Suppression of mouse lymphocyte responses to mitogens in vitro by Trypanosoma cruzi. International Journal for Parasitology14: 45–52. The ability of T. cruzi to inhibit mitogen-induced mouse lymphocyte responses was studied to find out if the organism itself is involved in the production of the immunosuppression that occurs during the acute phase of Chagas' disease. Significant suppression of normal spleen cell responses to concanavalin A (a T cell-specific mitogen) or to bacterial lipopolysaccharide (a B cell-specific mitogen) were seen when the concentration of either trypomastigote or epimastigote forms of the parasite reached or exceeded 2.5 × 10⁶ organisms/ml in the cultures. The inhibitory effect was noted over wide ranges of concentrations of either mitogen. Since spleen cells stimulated with mitogenic solutions that had been absorbed with 1 × 10⁷ parasites/ml produced significant responses, the suppressive effect could not be attributed just to mitogen removal by the parasites. Preparations of T. cruzi disrupted by freezing and thawing also inhibited mitogen-induced responses. This indicated that production of suppression was not a result of parasite competition for essential medium nutrients and that trypanosome viability was not required. Suppression was demonstrable only when the parasites were incorporated into the cultures within 12 h after mitogenic stimulation. These results taken together indicate that T. cruzi has the ability to modulate directly or indirectly lymphocyte function by interfering with the initial stages of commitment to lymphoproliferation.     

Evolution of infection in mice inoculated by the oral route with different developmental forms of Trypanosoma cruzi I and II

Oral infection has become the most important transmission mechanism of Chagas disease in Brazil. For this study, the development of Trypanosoma cruzi infection in mice, induced by the oral and intraperitoneal (IP) routes, was compared. Four groups of Swiss mice were used to evaluate the influence of parasite genetics, number of parasites, inoculation volume and developmental stages on the development of the orally induced infection: 1 – blood trypomastigotes (BT) via oral; 2 – BT via IP; 3 – culture metacyclic trypomastigotes (MT) via oral; and 4 – culture MT via IP. Animals inoculated orally showed levels of parasitemia, as well as infectivity and mortality rates, lower than animals inoculated via IP, regardless of DTU (discrete typing unit) and inoculum. Animals infected with TcII showed higher levels of these parameters than did animals infected with TcI. The larger volume of inoculum showed a greater capacity to cause an infection when administered via the oral route. BT infection was more virulent than culture MT infection for both routes (oral and IP). However, mice inoculated orally with BT showed lower levels than via IP, while mice inoculated orally with culture MT showed similar levels of infection to those inoculated via IP. Mice inoculated with culture MT showed more histopathological changes than those inoculated with BT, regardless of the inoculation route. These results indicate that this alternative experimental model is useful for evaluating infection by T. cruzi isolates with subpatent parasitemia and low virulence, such as those belonging to the TcI and TcIV DTUs, which are prevalent in outbreaks of orally transmitted Chagas disease.     

Eimeria stiedae: experimental infection in rabbits and the effect of treatment with toltrazuril and ivermectin

The aim of this study was to investigate the clinical, haematological, biochemical, lipid peroxidation, ultrasonographic and pathologic findings in hepatic coccidiosis induced by Eimeria stiedae in rabbits, and also to compare the treatment effects of both toltrazuril and ivermectin separately and in combination. In this study, 56 rabbits were divided into eight groups. The first group was designated as healthy control group. Rabbits were infected with 40.000 sporulated oocysts of E. stiedae. Groups 2, 3, 4, 5, 6, 7 and 8 were allocated as the infected control group, infected+toltrazuril-treated group, infected+ivermectin-treated group, infected+toltrazuril+ivermectin-treated group, non-infected+toltrazuril-treated group, non-infected+ivermectin-treated group, non-infected+toltrazuril+ivermectin-treated group, respectively. Haematocrit, Haemoglobin and MCV values as well as percentage of lymphocyte decreased in Groups 2 and 4 whereas leucocyte counts and percentage of granulocyte leucocyte increased. Serum GGT, ALT and AST activities increased but albumin value decreased. Plasma MDA concentrations increased whereas erythrocyte CAT, GSH-Px, and SOD activities decreased. Mean oocyst numbers in per gram faeces (epg values) increased in both groups during the study. Ultrasonographic examination revealed that the liver was enlarged and had hyperechogenic parenchyma. Bile ducts were dilated and hyperechogenic and the gall bladder was dilated. The livers of these animals were enlarged and typical macroscopic and microscopic findings of coccidiosis were present. Treatment with toltrazuril and toltrazuril+ivermectin combination were highly effective in reducing faecal oocyst output in infected rabbits. Haematological, biochemical and lipid peroxidation parameters and, ultrasonographic findings of the liver were close to control values for Groups 3 and 5. Necropsy of these animals showed no visible lesions related to hepatic coccidiosis although a few oocysts were detected in the bile duct epithelial cells.