Virulent Pathotypes Research Articles

Vaccinal control of Marek's disease: Current challenges, and future strategies to maximize protection

Marek's disease is an economically important lymphoid neoplasm of chickens, caused by oncogenic strains of Marek's disease herpesvirus. The disease can be successfully controlled by vaccination with attenuated or non-pathogenic MDV strains. However, vaccine failures do occur as field strains continue to evolve towards pathotypes of greater virulence, and this evolution is likely to be driven by the vaccines themselves. Two general strategies can be considered to improve protection by vaccination. Firstly by the development of novel vaccines, and secondly by maximizing the potential of existing vaccines. This second goal requires investigation of optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. Accurate quantitation of Marek's disease vaccine virus in vaccinated chicks will contribute significantly to our understanding of vaccinal protection. We recently developed a real-time polymerase chain reaction (PCR) assay for quantitation of CVI988 vaccine virus in the feather tips, a rich source of viral DNA which can easily be sampled in a non-invasive manner. This PCR test is now used commercially to confirm the successful vaccination of chicks. We have also used the PCR to examine various aspects of vaccination in experimental chicks and commercial chicks with a view to determining how vaccine level in feathers correlates with protection against challenge, and for identifying optimal timing and vaccine delivery route, and optimal vaccination regimes for different breeds of chick. In this article we review some aspects of the current vaccinal control of Marek's disease, before highlighting some of the problems associated with current vaccines and vaccination strategies, and the challenges for the future. We go on to discuss the development and use of our real-time PCR feather test, its current applications and potential opportunities in Marek's disease vaccine research.

Modulation of reactive oxygen species activities and H2O2 accumulation during compatible and incompatible tomato–root‐knot nematode interactions

Here, the interaction of Melodoigyne incognita virulent and avirulent pathotypes with susceptible and Mi-resistant tomato (Solanum lycopersicon) has been studied. Significant differences in nematode penetration occurred 2 days postinoculation (dpi) and became stable from 3 dpi onwards. The hypersensitive cell response (HR) in resistant plants prevented the installation of the avirulent pathotype. The virulent pathotype overcame the Mi (nematode) resistance and induced feeding sites in root cells without triggering HR. Reactive oxygen species (ROS), visualized by subcellular reduction of nitroblue tetrazolium, accumulated in nematode penetrated cells. Quantitative analyses with dichlorofluorescein indicated that the oxidative burst occurred very early with both pathotypes, with an enhanced rate in hyper-responsive cells. Hydrogen peroxide (H(2)O(2)), detected by cerium chloride reaction, accumulated in the cell walls and especially in cells neighbouring HR. The apoplastic location of cerium perhydroxide indicated that either the plasma membrane or the cell wall was the primary site of the superoxide/H(2)O(2) generator. The data provide evidence, for the first time, for ROS-generated signals and their spatiotemporal expression in the host and nonhost interaction of tomato with nematodes.

Examination of the Effect of a Naturally Occurring Mutation in Glycoprotein L on Marek's Disease Virus Pathogenesis

We recently reported a comparison of glycoprotein-encoding genes of different Marek's disease virus pathotypes (MDVs). One mutation found predominantly in very virulent (vv)+MDVs was a 12-bp (four-amino acid) deletion in the glycoprotein L (gL)-encoding gene in four of 23 MDV strains examined (three were vv+MDVs and one was a vvMDV). This mutation was noted in the gL of the TK (615K) strain, but not in the RL (615J) strain of MDV. These strains have identical mutations in the meq gene characteristic of vv+MDVs but can be distinguished by the mutation in the gL-encoding gene. The TK strain was originally isolated from vaccinated chickens and appeared to confer or enhance horizontal transmission of the vaccine virus, herpesvirus of turkeys (HVT). Because the molecular basis for increased virulence of MDV field strains is unknown, we hypothesized that one mechanism might be by coreplication of MDV-1 strains with HVT and that it could be mediated by the mutation of gL, an essential component of the glycoprotein H/L complex. In this study, we compared the pathogenicity of TK (615K) and RL (615J) strains of MDV in the presence and absence of simultaneous HVT coinfection. MDV infections were monitored at the levels of viremia (for both MDV-1 and HVT), clinical signs of MD, tumor incidence, and mortality in 1) inoculated chickens, 2) chickens exposed at 1 day of age, 3) chickens exposed at 2 wk of age, and 4) chickens exposed to both TK/HVT- and RL/HVT-infected chickens at 6 wk of age. We found high incidences of clinical MD signs in all inoculated treatment groups and all chickens exposed to TK and RL viruses, regardless of the presence of HVT. The median time to death of chickens exposed to TK1HVT-infected chickens, however, was lower than the other treatment groups for contact-exposed chickens. Although this difference was not considered to be statistically significant to a rigorously interpreted degree because of the removal of chickens for sampling from the test groups, these data suggest that replication of the TK strain and HVT, when coadministered, might incrementally affect the virulence of MDV-1 strains. The strict correlation of this enhancement of virulence with the mutation in gL, however, requires additional experiments with genetically identical MDV background strains.

Induction of a cytosolic pyruvate kinase 1 gene during the resistance response to Tobacco mosaic virus in Capsicum annuum

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P(0). Previously, to elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P(0) and genes specifically up-regulated during the HR were isolated by microarray analysis. One of the clones, Capsicum annuum cytosolic pyruvate kinase 1 (CaPK(c)1) gene was increased specifically in the incompatible interaction with TMV-P(0). The expression of CaPK(c)1 gene was also triggered not only by various hormones such as salicylic acid (SA), ethylene, and methyl jasmonate (MeJA), but also NaCl and wounding. These results suggest that CaPK(c)1 responds to several defense-related abiotic stresses in addition to TMV infection.

Exposure to Host Resistance Mechanisms Drives Evolution of Bacterial Virulence in Plants

Bacterial pathogenicity to plants and animals has evolved through an arms race of attack and defense. Key players are bacterial effector proteins, which are delivered through the type III secretion system and suppress basal defenses . In plants, varietal resistance to disease is based on recognition of effectors by the products of resistance (R) genes . When recognized, the effector or in this scenario, avirulence (Avr) protein triggers the hypersensitive resistance reaction (HR), which generates antimicrobial conditions . Unfortunately, such gene-for-gene-based resistance commonly fails because of the emergence of virulent strains of the pathogen that no longer trigger the HR . We have followed the emergence of a new virulent pathotype of the halo-blight pathogen Pseudomonas syringae pv. phaseolicola within leaves of a resistant variety of bean. Exposure to the HR led to the selection of strains lacking the avirulence (effector) gene avrPphB (or hopAR1), which triggers defense in varieties with the matching R3 resistance gene. Loss of avrPphB was through deletion of a 106 kb genomic island (PPHGI-1) that shares features with integrative and conjugative elements (ICElands) and also pathogenicity islands (PAIs) in diverse bacteria . We provide a molecular explanation of how exposure to resistance mechanisms in plants drives the evolution of new virulent forms of pathogens.

Development and validation of molecular markers linked to anAegilops umbellulata–derived leaf-rust-resistance gene,Lr9, for marker-assisted selection in bread wheat

An Aegilops umbellulata-derived leaf-rust-resistance gene, Lr9, was tagged with 3 random amplified polymorphic DNA (RAPD) markers, which mapped within 1.8 cM of gene Lr9 located on chromosome 6BL of wheat. The markers were identified in an F2 population segregating for leaf-rust resistance, which was generated from a cross between 2 near-isogenic lines that differed in the alien gene Lr9 in a widely adopted agronomic background of cultivar 'HD 2329'. Disease phenotyping was done in controlled environmental conditions by inoculating the population with the most virulent pathotype, 121 R63-1 of Puccinia triticina. One RAPD marker, S5550, located at a distance of 0.8+/-0.008 cM from the Lr9 locus, was converted to sequence-characterized amplified region (SCAR) marker SCS5550. The SCAR marker was validated for its specificity to gene Lr9 against 44 of the 50 known Lr genes and 10 wheat cultivars possessing the gene Lr9. Marker SCS5550 was used with another SCAR marker, SCS73719, previously identified as being linked to gene Lr24 on a segregating F2 population to select for genes Lr9 and Lr24, respectively, demonstrating the utility of the 2 markers in marker-assisted gene pyramiding for leaf-rust resistance in wheat.

Extracellular xylanases from two pathogenic races of Fusarium oxysporum f. sp. ciceris: enzyme production in culture and purification and characterization of a major isoform as an alkaline endo-β-(1,4)-xylanase of low molecular weight

Fusarium oxysporum f. sp. ciceris, the causal agent of Fusarium wilt of chickpea, comprises eight pathogenic races and two pathotypes. Races 0 and 5, representative of the least virulent yellowing pathotype and the most virulent wilt pathotype, respectively, produced extracellular xylanases when grown on minimal medium supplemented with either 1% commercial birchwood xylan or 0.3% chickpea cell walls. The pattern of extracellular proteins analysed by denaturing polyacrylamide gel electrophoresis in the two media presented some minor but distinctive differences between fungal races. By preparative isoelectrofocusing, the xylanase activity in cell wall-culture filtrates could be resolved into basic and neutral fractions with pI values around to 10 and 8, respectively, whereas the xylan-culture filtrates contained an additional acidic fraction of pI around 4. A common major xylanase was purified 7-fold to homogeneity by cation-exchange chromatography and chromatofocusing. The purified xylanase has a molecular weight of 21.6 kDa, optimum pH and temperature of 5.5 and 55 degrees C, respectively, pI in the range of 8.2 to 9.0, and Km and Vmax values of 2.24 mg ml(-1) (birchwood xylan as substrate) and 1200 nkat mg(-1) protein (72 U mg(-1) protein), respectively. The enzyme has an endo mode of action, hydrolysing xylan to xylobiose and higher short-chain xylooligosaccharides without forming free xylose.

CaAlaAT1 catalyzes the alanine: 2-oxoglutarate aminotransferase reaction during the resistance response against Tobacco mosaic virus in hot pepper

Hot pepper (Capsicum annuum L. cv. Bugang) plants exhibit a hypersensitive response (HR) upon infection by Tobacco mosaic virus (TMV) pathotype P0. To elucidate molecular mechanism that underlies this resistance, hot pepper cv. Bugang leaves were inoculated with TMV-P0 and genes specifically up-regulated during the HR were isolated by differential screening. One of the clones, CaAlaAT1 encoding a putative alanine aminotransferase (EC 2.6.1.2) exhibited organ-specific expression pattern and the transcript accumulated abundantly in red (ripe) fruit tissues. CaAlaAT1 transcript was also induced in older leaves during senescence. The expression of CaAlaAT1 gene was increased in the incompatible interaction with TMV-P0 but was not in the compatible interaction with TMV-P1.2. When a strain of Xanthomonas campestris pv. vesicatoria (Xcv) carrying an AvrBs2 gene was infiltrated into the leaves of a pepper cv. ECW 20R carrying Bs2 resistance gene, a marked induction and maintenance of CaAlaAT1 gene expression was observed. The expression of CaAlaAT1 gene was triggered by salicylic acid (SA) and ethylene but not by methyl jasmonate (MeJA). CaAlaAT1 seemed to be localized mostly at the cytosol from the polyethylene glycol (PEG)-mediated transformation experiment. CaAlaAT1 seemed to catalyze alanine: 2-oxoglutarate aminotransferase (AKT) reaction, which was a main activity among the four activities in vitro, during the resistance response against TMV in hot pepper. These results suggest that CaAlaAT1, a protein known to be involved in metabolic reactions, might be one of the components in the plant's defense signal pathway against pathogens.

Distribution and Characterization of Rice yellow mottle virus: A Threat to African Farmers.

Africa produces only 2.7% of the world’s rice and is the second-largest riceimporting region in the world (6.5 Mt in 2003). This amount represents about 25% of the world rice importation (40). With an average of 2 t/ha, excluding Egypt, rice production in Africa remains significantly below the world average (Asia 3.8 t/ha, Latin America 3.0 t/ha, United States 7.0 t/ha; Food and Agriculture Organization of the United Nations [FAO], published online). Insects and diseases are the two major constraints limiting rice production in Africa and Asia. Of all the rice diseases, the one caused by the Rice yellow mottle virus (RYMV), discovered in Kenya in 1970, is one of the most damaging in Africa. RYMV has been reported in many countries in East and West Africa, where in some cases whole fields have been devastated. RYMV has only been reported from the African continent and is endemic in every country where it has been reported. RYMV is a member of the genus Sobemovirus and possesses all the characteristic biophysical and biological properties of the members of the genus. The major insect vectors are chrysomelids, which play an essential role in primary infection, while secondary infection is due mainly to plant-to-plant contact. The virus particles are stable in infected dried leaves. Limitations to the spread of the virus include restricted mobility of the insect vectors, a limited host range, and absence of seed transmission. Partially resistant and highly resistant varieties have been identified, but currently the disease is not controlled adequately, and its incidence is increasing significantly in Africa. Due to the importance of rice as a staple food, the scientific community is active in studying the virus and the resistant mechanisms in rice. At the Institut de Recherche pour le Developpement (IRD) and the International Laboratory for Tropical Agricultural Biotechnology (ILTAB), different tools, such as RYMV polyclonal and monoclonal antibodies, have been produced for RYMV detection; and a series of studies has been initiated for a better understanding of the virus–host interactions. The synthesis of an infectious clone, the sequencing of many isolates of the virus, and structural studies of virus particles permitted a study of the function of the different viral proteins and an increased understanding of the virus cycle. African virologists also have initiated studies on the epidemiology of the RYMV, mainly the virulence and variability of virus strains collected throughout Africa (36,45). The relationships between genetic variability of the virus and geographical distances have been studied (19). Currently, phytopathologists are working in the field to better assess the agronomic impact of the virus (36,61,62). Breeders and geneticists at IRD and West African Rice Development Association (WARDA) in Cote d’Ivoire are working together to identify and isolate natural resistance genes (6,41). The International Institute for Tropical Agriculture (IITA) in Nigeria undertook breeding programs in Africa. Molecular biologists are offering new ways of controlling the disease through production of transgenic rice plants (37,52). This article is intended to provide a review of the disease and give an update on recent research results. The disease’s impact on rice production in Africa, its distribution, symptomatology, and epidemiology, the physical and genetic characteristics of the virus, and resistance mechanisms and breeding efforts for resistance will be discussed.

Molecular mapping of Aegilops speltoides derived leaf rust resistance gene Lr28 in wheat

In a segregating homozygous F2 population of bread wheat involving a leaf rust resistance gene Lr28 derived from Aegilops speltoides, six randomly amplified polymorphic DNA (RAPD) markers, three each in coupling and repulsion phase were identified as linked to Lr28, mapped to a region spanning 32 cM including the locus. The F2 and F3 populations were studied in the phytotron challenged with the most virulent pathotype 77-5 of leaf rust. A coupling phase linked RAPD marker S464721 and a repulsion phase linked RAPD marker S326550 flanked the gene Lr28 by a distance of 2.4± 0.016 cM on either side. The flanking markers genetically worked as co-dominant markers when analyzed together after separate amplification in the F2 population by distinguishing the homozygotes from the heterozygotes and increased the efficiency of marker assisted selection by reducing the false positives and negatives. One of the three RAPD markers, S421640 was converted to locus specific SCAR marker SCS421640 which was further truncated by designing primers internal from both ends of the original RAPD amplicon to eliminate a non-specific amplification of nearly same size. The truncated polymorphic sequence characterized amplified region marker (TPSCAR) SCS421570 was 70 bp smaller, but resulted in a single band polymorphism specific to Lr28 resistance. The TPSCAR marker was validated for its specificity to the gene Lr28 in nine different genetic backgrounds and on 43 of the 50 Lr genes of both native and alien origin, suggesting the utility of the SCAR markers in pyramiding leaf rust resistance genes in wheat.

Identification of a Brassica juncea‐derived recessive gene conferring resistance to Leptosphaeria maculans in oilseed rape

AbstractScreening of 212 spring type Brassica napus lines carrying B genome chromosome additions and introgressions from B. nigra, B. juncea and B. carinata resulted in the identification of one line segregating for resistance to Leptosphaeria maculans (anamorph Phoma lingam) at the seedling (cotyledon) stage. This line was derived from an interspecific hybrid containing the B genome of B. juncea. Trypan blue staining of cotyledons from resistant individuals demonstrated a hypersensitive response which is delayed in plants with intermediate lesion size. Genetic analysis supported the hypothesis of a monogenic recessive inheritance of resistance. The resistance gene, termed rjlm2, is effective in spring and winter type oilseed rape backgrounds against all tested virulent pathotypes, including two isolates which have been shown to overcome two dominant (race‐specific) B genome‐derived resistance genes in B. napus.

Ornithine Decarboxylase Gene (CaODC1) is Specifically Induced during TMV-mediated but Salicylate-independent Resistant Response in Hot Pepper

A gene encoding putative ornithine decarboxylase (ODC) has been isolated by differential screening of a cDNA library from the resistant hot pepper (Capsicum annuum L.) inoculated with avirulent tobacco mosaic virus (TMV) pathotype P0. In hot pepper plants, transcripts of the CaODC1 (C. annuum ODC1) gene started to accumulate at 24 h post-inoculation of TMV-P0 and the signal was spread systemically. The transcript level of CaODC1 was increased rapidly in a hot pepper resistant to Xanthomonas campestris pv. vesicatoria (Xcv) but not in a susceptible hot pepper after inoculation. These results suggest possible role(s) for CaODC1 in plant defense against a broad range of pathogens including viruses and bacteria.

Traits of Soybean-Infecting Phytophthora Populations from Illinois Agricultural Fields

Phytophthora rot caused by Phytophthora sojae is a common and significant disease of soybean (Glycine max) in Illinois and throughout the Midwestern United States. The pathogenic characteristics of P. sojae populations in several Midwestern states have been reported recently, but pathogenicity and fungicide sensitivity traits of populations in Illinois were poorly understood. Isolates (n = 121) of soybean-infecting Phytophthora spp. were baited using susceptible cv. Sloan seedlings from soybean field soils with a history of seedling diseases in 24 counties across Illinois. The pathotype and race of isolates of P. sojae were characterized using 11 differential soybean cultivars in greenhouse tests using a hypocotyl inoculation method. Sensitivity to the fungicidal compounds metalaxyl and mefenoxam was tested with 63 isolates in vitro. Most (96%) of the Phytophthora isolates sampled from Illinois soybean fields were P. sojae, but 4% were an unidentified Phytophthora sp. as determined by phenotypic and genotypic traits. We present a preliminary description of another Phytophthora sp. from soybean fields in a restricted region of Illinois that is pathogenic and capable of killing soybean. Based on eight Rps gene differentials (Rps1a, 1b, 1c, 1d, 1k, 3a, 6, and 7 ), 22 virulence pathotypes of P. sojae were identified and 88% of all isolates were characterized to a defined race. The four most common races, which were 58% of all isolates, were races 1 (21%), 4 (15%), 33 (12%), and 28 (10%). Based on 11 differentials, (those noted above and Rps 2, 4, and 5), 31 virulence pathotypes were identified. The mean virulence complexities, which are the number of susceptible interactions on the sets of 8 and 11 Rps gene differentials, were 3.3 and 3.7, respectively. All isolates tested were sensitive to Apron XL, Allegiance, technical grade mefenoxam, and technical grade metalaxyl at 1.0 μg a.i./ml. The population of P. sojae is diverse and composed of multiple pathotypes and races in Illinois, and the results suggest that pathogen virulence partially explains poor performance of Phytophthora-resistant cultivars in many Illinois soybean fields.

Atividade de peroxidase e polifenoloxidase na resistência do feijão à antracnose

O objetivo deste trabalho foi avaliar a influência das enzimas peroxidase e polifenoloxidase na resistência à antracnose de quatro cultivares de feijão. Plântulas de feijão foram pulverizadas com ácido salicílico e com a raça delta de Colletotrichum lindemuthianum (fungo indutor) e submetidas à inoculação do patótipo virulento 33/95 de C. lindemuthianum três dias após a aplicação do fungo indutor e do ácido salicílico. Essas plantas foram avaliadas quanto à atividade enzimática e teores de fenóis, três dias após a aplicação do fungo indutor e cinco dias após a inoculação do patótipo virulento. Acréscimos nas atividades dessas enzimas foram maiores nos tratamentos com ácido salicílico e fungo indutor em todas as cultivares. Maiores estímulos nas atividades enzimáticas foram observados nas cultivares com maior resistência à doença. Constatou-se o aparecimento de uma isoperoxidase nos tratamentos com fungo indutor, ácido salicílico, após inoculação do patótipo virulento, e na testemunha, nas cultivares AB 136, Rio Tibagi e Macanudo. Houve correlação positiva entre as atividades da peroxidase e da polifenoloxidase, os teores de compostos fenólicos e a resistência à antracnose.

Intestinal yersiniosis. Clinical importance, epidemiology, diagnosis, and prevention

Yersinia enterocolitica and Y. pseudotuberculosis are causative agents of yersiniosis in humans and animals that have to be separated from Y. pestis, the causative agent of plague, representing a separate clinical and epidemiological entity. Intestinal yersiniosis may manifest in humans as (1) enteritis, (2) terminal ileitis, mesenteric lymphadenitis, or pseudoappendicitis, and (3) septicemia leading to focal abscesses in spleen and liver. The intestinal infection may be followed by reactive arthritis in a number of cases. Y. enterocolitica and Y. pseudotuberculosis are distributed worldwide but occur mainly in moderate or subtropical climates. The most important reservoirs are rodents, lagomorphs, and birds for Y. pseudotuberculosis and domestic animals, especially pigs, for Y. enterocolitica. All Y. pseudotuberculosis isolates may be considered as pathogenic whereas Y. enterocolitica strains can be subdivided into pathotypes of different virulence. The differentiation of pathotypes by determination of the biovar and demonstration of the 75-kb virulence plasmid is therefore of diagnostic importance. Preventive measures include avoidance of direct infection by contact with infected reservoir animals and practice of good hygiene during slaughtering as well as in food production and preparation of meals.

Molecular characterization of pepper germin-like protein as the novel PR-16 family of pathogenesis-related proteins isolated during the resistance response to viral and bacterial infection.

To understand the molecular defense mechanism controlling the hypersensitive response (HR) better, we examined the hot pepper plant (Capsicum annuum L. cv. Bugang), which exhibits an HR in response to infection by Tobacco mosaic virus pathotype P0 (TMV-P0). A full-length cDNA clone was isolated by differential screening of a cDNA library that was constructed with mRNA extracted from hot pepper leaves during the resistance response to TMV-P0. The predicted amino acid sequence of the cDNA clone exhibited a high sequence similarity to germin-like protein (GLP). The CaGLP1 (Capsicum annuum GLP1) cDNA contains a single open reading frame of 660 bp encoding 220 amino acid residues. Upon inoculation with TMV or Xanthomonas, CaGLP1 transcripts were specifically accumulated in the incompatible interaction but not in the compatible interaction. In plants treated with salicylic acid (SA) or ethephon, which are signal molecules in the defense-related signal transduction pathway, CaGLP1 transcripts were accumulated rapidly. As far as we know, this is the first report that plant GLPs can be specifically induced during a defense response against viral infection. These data suggest that in the hot pepper plant CaGLP1 may be involved in the defense response to viral pathogens, and thus be classified as a new family of PR proteins, 'PR-16'.

Genetic Variation and Virulence on Lr26 in Puccinia triticina

ABSTRACT The genetic relationships between isolates of Puccinia triticina virulent on wheat with the Lr26 resistance gene were studied. The diversity within and between isolates of P. triticina from Israel, Europe, and the United States was determined by virulence on near-isogenic Thatcher lines and by random amplified polymorphic DNA. According to the molecular markers, isolates that were virulent on Lr26 had diversity levels similar to those of Lr26 nonpathogenic isolates. Distances between subpopulations of isolates virulent and avirulent on Lr26 varied and were unrelated to the Lr26 virulence phenotype. Cluster analysis suggested four groups, three of which were closely associated with the geographical origin of the isolates-Israel, the United States, and Europe. All four groups included both Lr26 virulent and avirulent pathotypes. The results showed that Lr26 virulent rust pathotypes are as genetically dissimilar as the rest of the population. The cluster analysis showed that the rust population in Israel includes at least two different subpopulations, both of which contain Lr26 virulent and Lr26 avirulent isolates.

Pathotyping of Newcastle disease virus with a filamentous bacteriophage.

A filamentous phage bearing the peptide sequence TLTTKLY was isolated from a heptapeptide phage display library against a velogenic Newcastle disease virus (NDV). In order to investigate the potential of this specific phage as an immunological reagent in virus pathotyping, an enzyme-linked immunosorbent assay (ELISA)-based method was developed. This method can differentiate the velogenic strains from the mesogenic and lentogenic strains. An equilibrium-binding assay in solution showed that the interactions between the phage and all the NDV strains gave rise to two widely differing dissociation constants (Kdrel). Based upon the first Kdrel values, NDV strains can be classified into two groups; the first comprises the velogenic strains, and the second consists of the mesogenic and lentogenic strains. These results indicate a high degree of correlation between the binding affinities and pathotyping of NDV strains using the TLTTKLY phage.

Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway.

A hot pepper (Capsicum annuum) cDNA clone encoding pathogenesis-related protein 10 (CaPR-10) was isolated by differential screening of a cDNA library prepared from pepper leaves inoculated with tobacco mosaic virus pathotype (TMV-P0). CaPR-10 transcripts were induced in the incompatible interaction with TMV-P0 or Xanthomonas campestris pv. vesicatoria (Xcv) but not induced in the compatible interaction. Characterization of enzymatic properties of CaPR-10 indicated that the recombinant protein exhibits a ribonucleolytic activity against TMV RNA, as well as against pepper total RNA, and shows its putative antiviral activity in several conditions. The CaPR-10 protein existed at very low level in leaf tissue but was dramatically induced as soon as plants were inoculated with TMV-P0, and this was correlated with the increase of its ribonucleolytic activity. Immunoblot analysis and pull-down assays using proteins extracted from pepper leaves showed that TMV-P0 inoculation led to the phosphorylation of CaPR-10, a modification that should affect its capacity for RNase function. We present data that the induction and subsequent phosphorylation of CaPR-10 increased its ribonucleolytic activity to cleave invading viral RNAs, and this activity should be important to its antiviral pathway during viral attack in vivo.

Induction of chalcone synthase and phenylalanine ammonia-lyase by salicylic acid and Colletotrichum lindemuthianum in common bean

The activities of the enzymes chalcone synthase (CHS) and phenylalanine ammonia-lyase (PAL) were measured in leaf extracts obtained from four cultivars of the common bean (AB 136, Rio Tibagi, Carioca and Macanudo). Two stages of plant development were examined: plantlets (V2) and the onset of blooming (R6). Initially, the plants were either treated with salicylic acid or inoculated with the delta race of Colletotrichum lindemuthianum (inductive fungus) and after three days they were evaluated for enzyme activity. Afterwards, all plants were inoculated (challenged) with the virulent pathotype 33/95 of C. lindemuthianum except for the water control. Five days later, the activities of PAL and CHS were evaluated. There were significant changes in the activities of both enzymes three days after treatment with salicylic acid or inductive fungus when compared to the control. Five days after inoculation with with the virulent pathotype 33/95 of C. lindemuthianum CHS activity in the Macanudo was similar to control plants that were not treated with salicylic acid or the inductive fungus but inoculated with 33/95 C. lindemuthianum. The increase in enzyme activity after challenge with 33/95 C. lindemuthianum was greatest for the salicylic acid treatment in the cultivar AB 136, followed by Rio Tibagi and Carioca.