Pathogenesis-related proteins in lima bean leaves infected with tobacco ringspot virus and their distribution within and around local lesions

Tobacco ringspot virus (TRSV) induces circular, darkbrown local lesions on primary leaves of lima bean (Phaseolus lunatus cv Nemagreen) with a concomitant production of three basic and three acidic pathogenesisrelated (PR) proteins. The three basic proteins are: a 21 kDa protein related serologically to Pinto bean PR-4d and tobacco PR-5 proteins; a 36 kDa glucanase that is related to tobacco PR-2; and, a 31 kDa chitinase related serologically to ethylene-induced bean chitinase. The three acidic 18 kDa lima bean PR proteins are serologically similar and probably are charged isomers of the same protein. The 21 kDa basic protein and the 18 kDa acidic protein accumulated preferentially at the lesion center while the 31 kDa chitinase and TRSV were distributed evenly throughout the necrotic area. In green tissue immediately surrounding a lesion, the amounts of PR proteins were comparable to or lower than those in the necrotic area, and virions were not detected. This mode of spatial distribution indicates that lima bean PR proteins are not involved in TRSV localization, and is consistent with other observations that PR proteins play no direct role in restricting viral spread.     

Homology between chitinases that are induced by TMV infection of tobacco

Recently, four chitinases have been detected in tobacco mosaic virus (TMV) infected tobacco: two acidic chitinases that were identified as pathogenesis-related (PR) proteins P and Q and two basic chitinases (Legrand et al., Proc.Natl. Acad. Sci. USA, in press). Here, it was shown that P and Q are closely serologically related but not related to other known acidic tobacco PR proteins. Antisera to P and Q were used to characterize translation products of TMV-induced mRNAs that were hybrid-selected with cDNA clones described previously (Hooft van Huijsduijnen et al., EMBO J 5: 2057–2061, 1986). In this way cDNA clones corresponding to the acidic and basic chitinases were identified. The partial amino acid sequences of the acidic and basic tobacco chitinases that were represented in the clones, showed an approximately 70% homology to each other and to the sequence of a bean chitinase. Although the acidic and basic chitinases differ in apparent molecular weight, they were found to have homologous C-termini.Hybridization of cDNA probes to genomic blots indicated that the acidic and basic chitinases are each encoded by two to four genes in the amphidiploid genome of Samsun NN tobacco. A similar complexity was found for the genes encoding the tobacco PR protein that is homologous to the sweet-tasting protein thaumatin and to the bifunctional trypsin/-amylase inhibitor from maize.     

Differential induction of acquired resistance and PR gene expression in tobacco by virus infection,ethephon treatment,UV light and wounding

Genes for acidic, extracellular and basic, intracellular pathogenesis-related (PR) proteins of tobacco were studied for their response to tobacco mosaic virus (TMV) infection, ethephon treatment, wounding and UV light. The genes encoding the acidic PR proteins (PR-1, PR-2, PR-3, PR-4 and PR-5) responded similarly to the different forms of stress. They appeared to be highly inducible by TMV, moderately inducible by ethephon treatment and UV light and not inducible by wounding. The genes for the basic counterparts of PR-1, PR-2, PR-3 and PR-5 also displayed a common stress response. However, this response was different from that of the acidic PR proteins. Here, the highest induction was obtained upon ethephon treatment, while the other stress conditions resulted in somewhat lower levels of expression. Most genes for acidic PR proteins are systemically induced in the uninfected upper leaves of TMV-infected plants, whereas the genes encoding the basic PR proteins are not. Increased levels of resistance to TMV, comparable to resistance obtained by pre-infection with the virus, were found in UV-irradiated leaves but not in wounded or ethephon-treated leaves. This indicates that the basic PR proteins are not involved in resistance to TMV infection. Tobacco phenylalanine ammonia-lyase genes were not inducible by the various stress conditions. The implications of these findings in relation to the phenomenon of acquired resistance are discussed.     

Spermine Is a Salicylate-Independent Endogenous Inducer for Both Tobacco Acidic Pathogenesis-Related Proteins and Resistance against Tobacco Mosaic Virus Infection

Intercellular spaces are often the first sites invaded by pathogens. In the spaces of tobacco mosaic virus (TMV)-infected and necrotic lesion-forming tobacco (Nicotiana tabacum L.) leaves, we found that an inducer for acidic pathogenesis-related (PR) proteins was accumulated. The induction activity was recovered in gel-filtrated fractions of low molecular mass with a basic nature, into which authentic spermine (Spm) was eluted. We quantified polyamines in the intercellular spaces of the necrotic lesion-forming leaves and found 20-fold higher levels of free Spm than in healthy leaves. Among several polyamines tested, exogenously supplied Spm induced acidic PR-1 gene expression. Immunoblot analysis showed that Spm treatment increased not only acidic PR-1 but also acidic PR-2, PR-3, and PR-5 protein accumulation. Treatment of healthy tobacco leaves with salicylic acid (SA) caused no significant increase in the level of endogenous Spm, and Spm did not increase the level of endogenous SA, suggesting that induction of acidic PR proteins by Spm is independent of SA. The size of TMV-induced local lesions was reduced by Spm treatment. These results indicate that Spm accumulates outside of cells after lesion formation and induces both acidic PR proteins and resistance against TMV via a SA-independent signaling pathway.     

Identification and kinetics of accumulation of proteins induced by ethylene in bean abscission zones

A two-dimensional gel electrophoresis system that combines a cationic polyacrylamide gel electrophoresis at pH near neutrality with sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to analyze the spectrum of basic polypeptides that accumulate in bean (Phaseolus vulgaris) abscission zones after treatment with ethylene. Results showed that, as abscission progressed, at least seven basic proteins accumulated in the abscission zone prior to the accumulation of 9.5 cellulase. Six of the seven proteins correspond to pathogenesis-related (PR) proteins. Among them, two isoforms of β-1,3-glucanase and multiple isoforms of chitinase were identified. A 22 kilodalton polypeptide that accumulated to high levels was identified as a thaumatin-like protein by analysis of its N-terminal sequence (up to 20 amino acids) and its serological relationship with heterologous thaumatin antibodies. A 15 kilodalton polypeptide serologically related to PR P1 (p14) from tomato was identified as bean PR P1 (p14)-like protein. The kinetics of accumulation of glucanases, chitinases, thaumatin-like and PR P1 (p14)-like proteins during ethylene treatment were similar and they showed that PR proteins accumulated in abscission zones prior to the increase in 9.5 cellulase. Addition of indoleacetic acid, a potent inhibitor of abscission, reduced the accumulation of these proteins to a similar extent (60%). The synchronized accumulation of this set of PR proteins, early in the abscission process, may play a role in induced resistance to possible fungal attack after a plant part is shed. The seventh protein does not correspond to any previously characterized PR protein. This new 45 kilodalton polypeptide accumulated in abscission zones on exposure to ethylene concomitantly with the increase in 9.5 cellulase. Its N-terminal sequence (up to 15 amino acids) showed some homology with the amino terminal sequence of chitinase. Polyclonal antibodies against chitinase recognized the 45 kilodalton polypeptide, but polyclonal antibodies against the 45 kilodalton protein recognized chitinase weakly. When abscission was inhibited by addition of indoleacetic acid, the accumulation of the 45 kilodalton protein was strongly inhibited (80%). This result suggests that the 45 kilodalton polypeptide may play a more direct role in abscission.     

Soluble pathogenesis-related proteins ofCapsicum annuum leaves induced by cucumber mosaic virus infection

Eight major pathogenesis related (PR) proteins were found in a soluble extract of cucumber mosaic virus (CMV) infectedCapsicum annuum leaves. None of them was present in the soluble extract of sham-inoculated controls. The proteins were detected by two-dimensional polyacrylamide gel electrophoresis using (2D-PAGE)PhastSystem, native-PAGE in the first dimension and SDS-PAGE in the second. Two major acidic PR-proteins were identified on the basis of their relative molecular mass (M_r), PR1 of 15.3 kD and PR2 of 29 kD. Six proteins were basic and were identified as PRla of 15.9 kD, PRlb of 15.0 kD, PRlc of 15.9 kD, PR2 of 27.0 kD, PR3 of 36.3 kD and PR4 of 48.8 kD.     

Sobemovirus genome appears to encode a serine protease related to cysteine proteases of picornaviruses

A putative serine protease was identified among non-structural proteins of southern bean mosaic virus (SBMV) by sequence comparison with cellular and viral proteases. The predicted SBMV protease displayed a significant similarity to cysteine proteases of picornaviruses, providing a possible evolutionary link between the two enzyme classes. It is suggested that SBMV follows the general expression strategy characteristic of other positive-strand RNA viruses containing 5'-terminal covalently linked proteins (VPg), i.e. generation of functional proteins by polyprotein processing.     

Single step purification of potent antigenic protein from sparganum by gelatin-affinity chromatography

Out of many component proteins in crude saline extract of Spirometra mansoni plerocercoid (sparganum), 36 kDa and 29 kDa proteins were found to be the most antigenic and were already purified by immunoaffinity chromatography using monoclonal antibody as a ligand. In this study, a single step purification of these potent antigenic proteins of sparganum extract was investigated. When the crude saline extract was charged to gelatin-Sepharose 4B affinity column, 36 kDa and 29 kDa protein fractions were bound. SDS-polyacrylamide gel electrophoresis (PAGE) and SDS-PAGE/immunoblot confirmed that the bound protein to gelatin was serologically pure. When evaluated by ELISA with patients sera, the purified protein of 36 and 29 kDa also showed improved antigenicity.     

Increased Resistance to the Spread of Tobacco Mosaic Virus in Pinto Bean Leaves Caused by Sugar and Light

Ultraviolet light (UV) irradiation increased expansion of TMV lesions in detached Pinto bean primary leaves incubated in darkness. However, if after UV-irradiation the leaves were incubated in the light, no increase in lesion expansion occurred. The light effect was considered not to be due to photorepair of UV damaged DNA, since non-photorepairing treatments such as incubation in red light, or delayed exposure to white light after UV irradiation also prevented increase in lesion expansion. The effect of visible light in preventing TMV-lesion enlargement was shown to be related to photosynthetic energy supply to the host cell defense mechanism since incubation of infected leaves in the presence of the photosynthesis inhibitor 3-(3,4-dichlorophenyl)-l,l-dimethyl urea (DCMU) in light caused large lesions whether leaves were irradiated by UV or not. Supplying 0.1 M sucrose in the dark also inhibited lesion enlargement in UV-irradiated or nonirradiated leaves. Dinitrophenol (DNP) negated the sucrose effect in the dark. However, in light incubation, DNP did not induce large lesions indicating that DNP did not interfere with energy supply in the light. It is concluded that the Pinto bean leaf cells can use energy derived both from mitochondria and chloroplasts for building the resistance mechanism to virus spread. In this case, cellular resistance to virus spread seems to be correlated with callose deposition on the walls of noninfected cells adjacent to the necrotic cells. Energy supply in various forms will assist host cells in building the resistance mechanism as well as retarding senescence. Detachment, prolonged dark incubation, or exogenous supply of DNP led to accelerated senescence which in turn led to secondary enlargement of lesions. The cause of such secondary enlargement may be explained by starvation of cells and disappearance of callose.     

Differential Regulation of beta-1,3-Glucanase Messenger RNAs in Response to Pathogen Infection

The acidic, extracellular, glucan endo-1,3-β-glucosidases (EC 3.2.1.39; β-1,3-glucanases), pathogenesis-related proteins-2, -N, and -O (i.e. PR-2, PR-N, and PR-O) were purified from Nicotiana tabacum (tobacco) and their partial amino acid sequences determined. Based on these data, complementary DNA (cDNA) clones encoding the proteins were isolated. Additional cDNAs were isolated that encoded proteins approximately 90% identical with PR-2, PR-N, and PR-O. Although the proteins encoded by these cDNAs have not been identified, their deduced amino acid sequences have slightly basic or neutral calculated isoelectric points, as well as carboxy-terminal extensions. These physical characteristics are shared by the vacuolar form of β-1,3-glucanase and other vacuolar localized analogs of PR proteins, suggesting that the unidentified proteins may be similarly localized. A preliminary evolutionary model that separates the β-1,3-glucanase gene family from tobacco into at least five distinct subfamilies is proposed. The expression of β-1,3-glucanase messenger RNAs (mRNAs) in response to infection by tobacco mosaic virus was examined. Messages for the acidic glucanases were induced similarly to the mRNAs for other PR proteins. However, the basic glucanase showed a different response, suggesting that different isoforms are differentially regulated by tobacco mosaic virus infection at the mRNA level.     

Occurrence,distribution and seasonal changes of viruses infecting common bean in northwestern Iran

Many surveys were conducted during 2003–2005 to study the identity, prevalence and fluctuation of bean infecting viruses in northwestern Iran. In total, 649 bean samples with virus- like symptoms were collected and analysed by double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and tissue-print immunoassay to detect infectious viruses. Serological tests revealed the presence of Bean common mosaic virus (BCMV), Bean common mosaic necrosis virus (BCMNV), Bean yellow mosaic virus (BYMV), Cucumber mosaic virus (CMV), Alfalfa mosaic virus (AMV), Bean leaf roll virus (BLRV), Bean pod mottle virus (BPMV) and Southern bean mosaic virus (SBMV), with some co-infection occurred, with prevalence of BCMV, BCMNV and BYMV (17–29% infection rate). The incidence of viruses showed variation in over 3 years of research including more than double increase in CMV from 2004 to 2005 and obvious one-third decrease in AMV from 2003 to 2005. SBMV and BPMV were detected sporadically in the fields and the response of some differential test plants was analysed by mechanical inoculation. Western immunoblotting analysis of SBMV infected bean leaf total proteins using SBMV-specific polyclonal antibody revealed viral CP with molecular mass of 28.5 kDa which confirmed the presence of SBMV as a new threat for bean production.     

Increased Larval Growth and Preference for Virus-Infected Leaves by the Mexican Bean Beetle, Epilachna varivestis Mulsant, a Plant Virus Vector

Phaseolus vulgaris L. cv. 'Black Valentine' is a systemic host for the plant viruses Southern bean mosaic virus (SBMV) and bean pod mottle virus (BPMV). The Mexican bean beetle, Epilachna varivestis Mulsant, is a vector of SBMV and BPMV. Our objective was to determine if the interaction of SBMV and BPMV with 'Black Valentine' bean plants would affect beetle behavior and growth. In adult feeding preference test assays, beetles preferred and ingested more of the virus-infected bean leaf tissue than the noninfected leaf tissue. Beetle larvae that fed on SBMV- or BPMV-infected plants weighed more than those that fed on healthy plants. Our experiments suggest that there might be a mutually beneficial relationship between the beetle and the viruses that it vectors. The virus benefits from being transmitted and the beetle benefits from better larval growth when feeding on virus-infected leaf tissue. This study further demonstrates the complexity of relationships between multiple organisms.     

β-1,3-glucanase and chitinase as pathogenesis-related proteins in the defense reaction of twoCapsicum annuum cultivars infected with cucumber mosaic virus

Pathogenesis-related (PR) proteins from pepper (Capsicum annuum L.) cv. Americano (tolerant) and cv. Smith-5 (sensitive), both elicited by infection with cucumber mosaic virus (CMV), were assayed for chitinase and glucanase activities. Two basic PR-proteins, M_r 49.0 and 28.0 kD, were elicicited from the intracellular fraction (INTRA-F) of both cvs. by CMV infection, while four acidic M_r 15, 19, 36 and 40 kD and two basic M_r 21.2 and 24 kD PR-proteins were elicited from the intercellular fluid (IF) of cv. Americano leaves. Five acidic M_r 21.5, 23.2, 24.4, 25.2 and 36 kD and five basic M_r 23.3, 26, 28.8, 30 and 32.3 kD PR proteins were elicited from the IF of cv. Smith-5. Isoelectric focusing (IEF) of the IF and the INTRA-F proteins revealed the occurrence, in both pepper cultivars, of one acidic M_r 36 kD and one basic M_r 25 kD PR-protein with glucanase activity. After native-PAGE for acidic proteins, the acidic PR-protein of Rf 0.7 and M_r 36 kD present in the IF of both pepper cvs. showed glucanase activity. Native-PAGE for basic proteins of the INTRA-F showed the presence of one band (Rf 0.61, M_r 25 kD) common to both cvs. and two additional bands (Rf 0.49, M_r 26 kD and Rf 0.79, M_r 33 kD) in the cv. Americano with glucanase activity. The specificity shown by the basic PR-proteins suggests glucanase activity is involved in the mechanisms of resistance to CMV in the cv. Americano. There was no difference in chitinase isoform patterns between the two pepper cultivars analyzed. After IEF of the IF proteins, one acidic chitinase isoform was detected. Native-PAGE separation of the IF showed one band (M_r 30 kD) with chitinase activity. Chitinase activity was not detected in the INTRA-F of either cultivar.     

The Pathogenesis-Related Proteins of Phsseolus Bean

Seven acidic low molecular weight pathogenesis-related (PR) proteins from tobacco necrosis virus-infected Phaseolus bean leaves were isolated, purified and partially characterized. The proteins could ba classified following the nomenclature of Antoniw et al. (1980) for tobacco PR proteins. Three groups are thus proposed, corresponding to molecular sizes of 16, 27 and 31 kD, very close to the values of three groups proposed for tobacco. Tentative correspondences between the PR proteins here described and those previously reported by other authors are also proposed.     

A structural comparison of concanavalin a and tomato bushy stunt virus protein

Summary Significant structural equivalence has been found among the polypeptide folds of the two tomato bushy stunt virus (TBSV) subunit domains and concanavalin A. This suggests gene duplication in the TBSV coat protein and leads to speculation on common functional properties of concanavalin A and viral coat proteins.Non-standard abbreviations TBSV tomato bushy stunt virus - SBMV southern bean mosaic virus     

Identification and partial characterization of two cysteine proteases from castor bean leaves (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.) activated by wounding and methyl jasmonate stress

Jasmonates are signaling molecules that play key roles in wound response and regulate the biosynthesis of many defensive proteins, including proteases. In this study, we investigate the effects of wounding and methyl jasmonate (MJ) application on the protein expression pattern of Ricinus communis L. leaves and on proteolytic activity. Gelatin zymography demonstrated that both MJ and mechanical wounding induce alterations in the proteolytic pattern of castor bean leaves (R. communis L.). Expression of two cysteine proteases (38 and 29 kDa) was induced by the treatments employed; however, MJ induced a higher protease level than mechanical wounding during the stress period (24, 48, and 72 h). The increase in protease activity mirrors the decline in soluble protein content and rubisco degradation that may indicate initiation of senescence in castor plants. The 29 kDa protease has an acidic optimal pH; whereas the 38 kDa protease has a neutral optimum activity. Both proteases were almost completely inhibited by E-64 and cystatin. The significant induction of these proteins by MJ suggests a possible role of cysteine proteases in leaf senescence as well as their involvement in regulating both the wound response and MJ in castor bean plants.     

Purification and serological characterization of three basic 15-kilodalton pathogenesis-related proteins from tomato

In tomato (Lycopersicon esculentum) several acidic and basic apoplastic pathogenesis-related (PR) proteins are induced upon inoculation with virulent or avirulent races of Cladosporium fulvum (Cooke) (syn. Fulvia fulva [Cooke] Cif). One of the most predominant and best characterized tomato PR proteins is P14, a basic protein that shows homology to the tobacco (Nicotiana tabacum) PR-1 protein family. To investigate whether, by analogy with these tobacco PR-1 proteins, P14 also belongs to a family of differently charged isomers, the abundantly occurring PR proteins with molecular masses around 15 kilodaltons (kD) were purified from apoplastic fluids isolated from C. fulvum-infected tomato. Three basic proteins migrating similarly to P14 on sodium dodecyl sulfate polyacrylamide gels were purified to homogeneity by gel filtration followed by high resolution liquid chromatography. Two proteins (15.5 kD, isoelectric point [pl] 10.9 and 10.7 appeared to be serologically related to each other and to the tobacco PR-1 proteins. A third protein (15 kD, pl 10.4) was not serologically related to any other tomato PR protein but was found to be related to PR-R from tobacco.     

Propyl gallate,a free radical scavenger,counteracts the benefits of exogenously applied salicylic acid and aggravates the deleterious effects of the southern bean mosaic virus in Rhizobium-nodulated Phaseolus vulgaris plants

Infection of Rhizobium-nodulated Phaseolus vulgaris by the southern bean mosaic virus (SBMV) markedly decreased the growth and nodulation of plants. Exogenous applications of salicylic acid (SA) at concentrations ≥10 μM further decreased growth and nodulation of virus-infected (V) plants. Only SA concentration of 5 μM in the solution improved the growth, nodulation, chlorophyll concentration and the catabolism of ureide in leaves of V plants. The spray of leaves with 2 mM propyl gallate (+Pg) decreased growth, nodulation as well as the chlorophyll and leaf ureide concentrations in V plants, regardless of the concentration of SA at which plants were grown. Ultrastructural damages in leaf cells caused by SBMV were also enhanced in V+Pg plants. The massive proliferation of virus particles and the presence of virus crystalline arrays within symbiosomes of nodules in V+Pg plants were attributed to proliferation of branched plasmodesmata in leaf vascular-parenchyma cell walls that facilitated virus movement. Virus particles were never observed in leaf and nodule tissues of V plants not sprayed with Pg. Exogenous applications of SA hindered while Pg increased the symbiotic performance of H plants, pointing out the complexity to be addressed in breeding for virus resistance in Rhizobium-nodulated beans.     

Three major somatic embryogenesis related proteins in Cichorium identified as PR proteins

In Cichorium hybrid clone '474' (C. intybus L., var. sativum x C. endivia L., var. latifolia), the direct somatic embryogenesis process in leaf tissues is accompanied by an overall increase in the amount of proteins secreted into the culture medium. Amongst these, three major protein bands of 38 kDa, 32 kDa and 25 kDa were found in the conditioned media. These extracellular protein bands accumulated in the medium of the embryogenic Cichorium hybrid up to 8-fold compared with those in the medium of a nonembryogenic variety. 32 and 25 kDa proteins were purified from the medium and their identities were determined as already described for 38 kDa beta-1,3-glucanases. To investigate their possible function in somatic embryogenesis, peptide sequences, serological relationships or biochemical properties revealed that there were at least two acidic chitinases of 32 kDa and one glycosylated osmotin-like protein of 25 kDa in the embryogenic culture medium. Comparing the amounts of the 38 kDa glucanases, the 32 kDa chitinases, and the 25 kDa osmotin-like protein present in the conditioned media of the embryogenic '474' hybrid and of a non-embryogenic variety, a 2-8-fold higher accumulation of these proteins was observed in the embryogenic hybrid culture medium. This may suggest that part of the accumulation of these three pathogenesis-related (PR) proteins could be correlated with the somatic embryogenesis process. Their possible involvement in this developmental process is discussed.     

Sobemovirus RNA linked to VPg over a threonine residue

Positive sense ssRNA virus genomes from several genera have a viral protein genome-linked (VPg) attached over a phosphodiester bond to the 5' end of the genome. The VPgs of Southern bean mosaic virus (SBMV) and Ryegrass mottle virus (RGMoV) were purified from virions and analyzed by mass spectrometry. SBMV VPg was determined to be linked to RNA through a threonine residue at position one, whereas RGMoV VPg was linked to RNA through a serine also at the first position. In addition, we identified the termini of the corresponding VPgs and discovered three and seven phosphorylation sites in SBMV and RGMoV VPgs, respectively. This is the first report on the use of threonine for linking RNA to VPg.