In situ localization of PR-1 mRNA and PR-1 protein in compatible and incompatible interactions of pepper stems withPhytophthora capsici

Summary In situ hybridization and immunogold labeling were performed to examine the temporal and spatial expression pattern of pathogenesis-related protein 1 (CABPR1) mRNA and PR-1 protein in pepper (Capsicum annuum L.) stem tissues infected by virulent and avirulent isolates ofPhytophthora capsici. CABPR1 mRNA accumulation was confirmed in the infected pepper stem tissue by Northern blot analysis and in situ hybridization. Northern blot analysis showed that the temporal expression ofCABPR1 mRNA varied greatly between compatible and incompatible interactions. An earlier expression of theCABPR1 gene, 6 h after inoculation, was observed in the incompatible interaction. In situ hybridization results revealed thatCABPR1 mRNA was expressed in the phloem areas of vascular bundles in infected pepper stem tissues, but especially strongly in the incompatible interaction. PR-1 protein was predominantly found in the intercellular spaces of pepper stem cells in the compatible and incompatible interactions 24 h after inoculation. Strikingly, the immunogold labeling was associated with fibrillar and electron-dense material localized in the intercellular space. Dense labeling of PR-1 protein was also seen at the interface of the pathogen and the host cell wall, whereas few gold particles were detected over the host cytoplasm. However, PR-1 protein was not detected over the fungal cell wall in either interaction.     

Subcellular localization of β-1,3-glucanase in Puccinia recondita f.sp. tritici-infected wheat leaves

An antiserum raised against the purified 33-kDa β-1,3-glucanase of wheat (Triticum aestivum L.) was employed to investigate the ultrastructural localization of the enzyme in wheat leaves infected with Puccinia recondita Rob. ex Desm. f.sp. tritici Eriks. and Henn. using a post-embedding immunogold labelling technique. In both compatible and incompatible interactions, β-1,3-glucanase was detected in the host plasmalemma and in the domain of the host cell wall near the plasmalemma of the mesophyll cells, but higher concentrations of the enzyme were detected in infected resistant wheat leaves than in infected susceptible ones. β-1,3-Glucanase was also found in the secondary thickening of xylem vessels and in the walls of guard cells, epidermal cells and phloem elements, while no labelling was observed in host organelles, viz. vacuoles, mitochondria, endoplasmic reticulum, Golgi bodies, nuclei and chloroplasts. A low concentration of the enzyme was detected on the intercellular hyphal wall and in the hyphal cytoplasm. In the compatible interaction, β-1,3-glucanase was demonstrated to accumulate predominantly in the haustorial wall and extrahaustorial matrix. In the incompatible interaction, strong labelling for β-1,3-glucanase was found in host cell wall appositions, in the extracellular matrix in the intercellular space, and in electron-dense structures of host origin which occurred in the incompatible interaction only. Received: 22 July 1997 / Accepted: 16 August 1997     

Rapid induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glycine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis

The differential regulation of the activities and amounts of mRNAs for two enzymes involved in isoflavonoid phytoalexin biosynthesis in soybean was studied during the early stages after inoculation of primary roots with zoospores from either race 1 (incompatible, host resistant) or race 3 (compatible, host susceptible) of Phytophthora megasperma f.sp. glycinea, the causal fungus of root rot disease. In the incompatible interaction, cloned cDNAs were used to demonstrate that the amounts of phenylalanine ammonia-lyase and chalcone synthase mRNAs increased rapidly at the time of penetration of fungal germ tubes into epidermal cell layers (1–2 h after inoculation) concomitant with the onset of phytoalxxin accumulation; highest levels were reached after about 7 h. In the compatible interaction, only a slight early enhancement of mRNA levels was found and no further increase occurred until about 9 h after inoculation. The time course for changes in the activity of chalcone synthase mRNA also showed major differences between the incompatible and compatible interaction. The observed kinetics for the stimulation of mRNA expression related to phytoalexin synthesis in soybean roots lends further support to the hypothesis that phytoalexin production is an early defense response in the incompatible plant-fungus interaction. The kinetics for the enhancement of mRNA expression after treatment of soybean cell suspension cultures with a glucan elicitor derived from P. megasperma cell walls was similar to that measured during the early stages of the resistant response of soybean roots.Abbreviations cDNA copy DNA - CHS chalcone synthase - PAL phenylalanine ammonia-lyase     

Proteomic analysis of pathogenesis-related proteins (PRs) induced by compatible and incompatible interactions of pepper mild mottle virus (PMMoV) in Capsicum chinense L3 plants

Resistance conferred by the L³ gene is active against most ofthe tobamoviruses, including the Spanish strain (PMMoV-S), aP_1,2 pathotype, but not against certain strains of pepper mildmottle virus (PMMoV), termed P_1,2,3 pathotype, such as the Italianstrain (PMMoV-I). Both viruses are nearly identical at theirnucleotide sequence level (98%) and were used to challenge Capsicumchinense PI159236 plants harbouring the L³ gene in order tocarry out a comparative proteomic analysis of PR proteins inducedin this host in response to infection by either PMMoV-S or PMMoV-I.PMMoV-S induces a hypersensitive reaction (HR) in C. chinensePI159236 plant leaves with the formation of necrotic local lesionsand restriction of the virus at the primary infection sites.In this paper, C. chinense PR protein isoforms belonging tothe PR-1, β-1,3-glucanases (PR-2), chitinases (PR-3), osmotin-likeprotein (PR-5), peroxidases (PR-9), germin-like protein (PR-16),and PRp27 (PR-17) have been identified. Three of these PR proteinisoforms were specifically induced during PMMoV-S-activationof C. chinense L³ gene-mediated resistance: an acidic β-1,3-glucanaseisoform (PR-2) (M_r 44.6; pI 5.1), an osmotin-like protein (PR-5)(M_r 26.8; pI 7.5), and a basic PR-1 protein isoform (M_r 18;pI 9.4–10.0). In addition, evidence is presented for adifferential accumulation of C. chinense PR proteins and mRNAsin the compatible (PMMoV-I)–C. chinense and incompatible(PMMoV-S)–C. chinense interactions for proteins belongingto all PR proteins detected. Except for an acidic chitinase(PR-3) (M_r 30.2; pI 5.0), an earlier and higher accumulationof PR proteins and mRNAs was detected in plants associated withHR induction. Furthermore, the accumulation rates of PR proteinsand mRNA did not correlate with maximal accumulation levelsof viral RNA, thus indicating that PR protein expression mayreflect the physiological status of the plant. Key words: Capsicum chinense, compatible interaction, incompatible interaction, HR-induction, PMMoV, PR proteins Received 5 December 2007; Revised 21 January 2008 Accepted 22 January 2008     

Immunogold localization of THRGP-like epitopes in the haustorial interface of obligate,biotrophic fungi on monocots

Summary Immunoelectron microscopy was used to determine the subcellular distribution of threonine-hydroxyproline-rich glycoprotein (THRGP) epitopes in host-parasite interactions between obligate, biotrophic fungi and cereals. Infection sites of stem rust (Puccinia graminis f. sp.tritici) and leaf rust (Puccinia recondita) on primary leaves of wheat (Triticum aestivum), as well as of powdery mildew (Erysiphe graminis f. sp.hordei) on coleoptiles of barley (Hordeum vulgare), wete probed with a polyclonal antiserum to maize THRGP. A few immunogold particles were found over the cell walls of wheat mesophyll tissue and barley coleoptile epidermis. Unlike previous examples in dicot plants, no enhanced accumulation of THRGP was observed in cereal cell walls adjacent to sites of pathogen ingress. Instead, the most pronounced accumulation of THRGP-like molecules occurred over the extrahaustorial matrix in both incompatible and compatible plant-pathogen interactions. For powdery mildew of barley, immunogold staining was distinctly increased over the center of the penetration sites; however, no labeling was found over papillae that formed during incompatible and compatible interactions. In addition, no cross-reactivity of the anti-THRGP antiserum with intercellularly growing rust pathogens was observed. The highly localized deposition of THRGP-like molecules in the extrahaustorial matrix suggests that the host plant establishes a modified barrier between itself and the pathogen.Abbreviations C chloroplast - EC plant epidermal cell - EM extrahaustorial membrane - EMA extrahaustorial matrix - GO Golgi body - GRP glycine-rich protein - HP high pressure - HRGP hydroxyprolinerich glycoprotein - Hyp hydroxyproline - LT low temperature - PBS phosphate-buffered saline - PBST PBS with Tween-20 - THRGP threonine-hydroxyproline-rich glycoprotein - VA vesicular arbuscular     

Purification of p47f,a Necrosis‐Inducing Protein Fraction from the Secretome of Phytophthora capsici

The oomycete Phytophthora capsici causes wilting disease in chilli pepper and another solanaceous plants, with important economic consequences. Although much investigation has been conducted about this pathogen, little is still known about which of its proteins are involved in the infection process. In this study, the bioassay‐guided fractionation of the secretome of P. capsici resulted in the purification of a phytotoxic protein fraction designated as p47f, capable of inducing wilting and necrosis on leaves of Capsicum chinense Jacq, and having a 47 kDa polypeptide with proteolytic activity as the major component. The isolated p47f fraction induced DNA degradation and decreased cell survival of C. chinense cell suspension culture. Sequencing of p47f indicated the presence of 15 proteins, which could be grouped into seven classes including a protease group, cell wall remodelling proteins and the transglutaminase elicitor M81D, among others. This is the first report of P. capsici secreting proteins that modulate cell responses mediated by ROS in the host.     

Molecular Characterization of an Oomycete-Responsive PR-5 Protein Gene from <Emphasis Type="Italic">Zingiber zerumbet</Emphasis>

The tropical spice crop ginger (Zingiber officinale Roscoe) is highly susceptible to soft rot disease caused by the necrotrophic oomycete Pythium aphanidermatum (Edson) Fitzp. However, Zingiber zerumbet (L.) Smith, a wild relative of ginger, is resistant to P. aphanidermatum and has been proposed as a potential donor for soft rot resistance to Z. officinale. We identified a member of the pathogenesis-related protein group 5 (PR-5) gene family in Z. zerumbet that is expressed constitutively but upregulated in response to infection by P. aphanidermatum. Expression of this gene was upregulated as early as 1.5 h post inoculation (hpi) with the pathogen, peaked at 6 hpi, declined by 9 hpi, and again peaked at 15 hpi before declining at 48 hpi. A cDNA of this PR-5 gene, designated as ZzPR5, encodes a 226-amino-acid predicted protein with a calculated pI of 5.05. The N terminus of this protein contains a 22-amino-acid signal peptide, suggesting that the protein may show apoplastic accumulation like other acidic PR-5 proteins. Phylogenetic analysis revealed high similarity between ZzPR5 and PR-5 proteins reported from other plant species, especially from other Zingiberales. Molecular modeling of ZzPR5 protein revealed an acidic surface cleft, a feature characteristic of glycoside hydrolases and antifungal PR-5 proteins. In molecular docking studies, a linear polymeric molecule of (1,3)-β-d-glucan, a major constituent of the oomycete cell wall, fitted favorably into the surface cleft of ZzPR5 and interacted with acidic amino acids known to be involved in glucan hydrolysis, suggesting a potential antioomycete activity for ZzPR5 protein. Elucidation of the molecular mechanism of ZzPR5 may provide important insight toward engineering soft rot resistance into the obligatory asexual ginger.     

Mass spectrometric identification of isoforms of PR proteins in xylem sap of fungus-infected tomato

The protein content of tomato (Lycopersicon esculentum) xylem sap was found to change dramatically upon infection with the vascular wilt fungus Fusarium oxysporum. Peptide mass fingerprinting and mass spectrometric sequencing were used to identify the most abundant proteins appearing during compatible or incompatible interactions. A new member of the PR-5 family was identified that accumulated early in both types of interaction. Other pathogenesis-related proteins appeared in compatible interactions only, concomitantly with disease development. This study demonstrates the feasibility of using proteomics for the identification of known and novel proteins in xylem sap, and provides insights into plant-pathogen interactions in vascular wilt diseases.     

Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.     

A novel Arabidopsis–oomycete pathosystem: differential interactions with Phytophthora capsici reveal a role for camalexin,indole glucosinolates and salicylic acid in defence

Phytophthora capsici causes devastating diseases on a broad range of plant species. To better understand the interaction with its host plants, knowledge obtained from a model pathosystem can be instrumental. Here, we describe the interaction between P. capsici and Arabidopsis and the exploitation of this novel pathosystem to assign metabolic pathways involved in defence against P. capsici. Inoculation assays on Arabidopsis accessions with different P. capsici isolates revealed interaction specificity among accession‐isolate combinations. In a compatible interaction, appressorium‐mediated penetration was followed by the formation of invasive hyphae, haustoria and sporangia in leaves and roots. In contrast, in an incompatible interaction, P. capsici infection elicited callose deposition, accumulation of active oxygen species and cell death, resulting in early pathogen encasement in leaves. Moreover, Arabidopsis mutants with defects in salicylic acid signalling, camalexin or indole glucosinolates biosynthesis pathways displayed severely compromised resistance to P. capsici. It is anticipated that this model pathosystem will facilitate the genetic dissection of complex traits responsible for resistance against P. capsici.     

Cloning,Expression, Purification and Initial Analysis of a Novel Pectate Lyase Pcpel1 from Phytophthora capsici

Phytophthora capsici inflicts damage on numerous crop plants by secreting a series of pectinase including pectate lyase (PEL). Here, we report a pectate lyase gene (Pcpel1) from a genomic library of a highly virulent P. capsici strain SD33. Pcpel1 was identified as an open reading frame of 1233 bp encoding a protein of 410 amino acids with a predicted amino‐terminal signal sequence of 21 amino acids. The predicted protein of Pcpel1 has a calculated molecular mass of 43.8 kDa and a pI value of 6.8. Analysis of the amino acid sequence suggested that it was a member of the polysaccharide lyase family 1 that shows pectate lyase activity. Moreover, heterologous expression of Pcpel1 in Pichia pastoris produced proteins with molecular mass 66 kDa, very likely due to differential glycosylation by the yeast. By western blotting and northern blotting analysis, Pcpel1 was strongly expressed during interaction of P. capsici with the host plant, suggesting its involvement in the process of host infection. The role of Pcpel1 in cell wall disassembly and host/parasite interaction is discussed.     

In vitro and in vivo antimicrobial activity of Xenorhabdus bovienii YL002 against Phytophthora capsici and Botrytis cinerea

Aims: Developing new bio‐agents to control plant disease is desirable. Entomopathogenic bacteria Xenorhabdus spp. have potential antimicrobial activity in agriculture. This work was conducted to evaluate the antimicrobial activity of Xenorhabdus bovienii YL002 on plant pathogenic fungi and oomycete in vitro and the efficiency of this strain to reduce the in vivo incidence of grey mould rot on tomato plants caused by Botrytis cinerea and leaf scorch on pepper plants caused by Phytophthora capsici. Methods and Results: The antimicrobial activity of X. bovienii YL002 was firstly determined on in vitro plant pathogenic fungi and oomycete and then on tomato fruits and plants infected with B. cinerea and pepper plants infected with P. capsici. The cell‐free filtrate of X. bovienii YL002 exhibited highest inhibition effects (>98%) on mycelia growth of P. capsici and B. cinerea. The 50% inhibition concentration (EC₅₀) of the methanol‐extracted bioactive compounds (methanol extract) of the cell‐free filtrate against P. capsici and B. cinerea were 164·83 and 42·16 μg ml^?1. The methanol extract also had a strong effect on the spore germination of P. capsici and B. cinerea, with a EC₅₀ of 70·38 and 69·33 μg ml^?1, respectively. At 1000 μg ml^?1, the methanol extract showed a therapeutic effect of 70·82% and a protective effect of 77·4% against B. cinerea on tomato plants compared with the control. The methanol extract also showed potent effect against P. capsici, with a therapeutic effect of 68·14% and a protective effect of 65·46% on pepper plants compared with the control. Conclusions: Xenorhabdus bovienii YL002 produces antimicrobial compounds with strong activity on plant pathogenic fungi and oomycete and has the potential for controlling grey mould rot of tomato plants and leaf scorch of pepper and could be useful in integrated control against diverse plant pathogenic fungi and oomycete. Significance and Impact of the Study: This study showed the potential that X. bovienii YL002 can be used to control the grey mould rot caused by B. cinerea on tomato plants and leaf scorch caused by P. capsici on pepper plants with the objective to reduce treatments with chemical fungicides.     

Increased expression of a plant actin gene during a biotrophic interaction between round-leaved mallow, Malva pusilla, and Colletotrichum gloeosporioides f. sp. malvae

Two actin genes, actA from the hemibiotrophic anthracnose fungus, Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. f. sp. malvae, and act1 from its host, Malva pusilla (Sm.) were cloned from a cDNA library developed from infected host tissue. The actin gene, actA, of C. gloeosporioides f. sp. malvae, which is similar to that of other euascomycetes, appears to be expressed constitutively. The actin gene of M. pusilla is most similar to one of the actin genes of Arabidopsis thaliana that is unique in being responsive to environmental stimuli such as wounding. Expression of actA was used to follow the growth of the fungus in the plant tissue. Low actA expression occurred until 72–96 h after inoculation and then increased rapidly, corresponding with the timing of the shift from slower biotrophic fungal growth to much more rapid necrotrophic growth. In contrast, expression of act1 approximately doubled during the biotrophic phase and then rapidly declined during the necrotrophic phase. Increased host actin expression could be due to host cytoskeleton rearrangement in response to biotrophic infection, and the subsequent decrease in host actin expression could be due to host cell disruption resulting from tissue maceration during necrosis. This is the first report of a host actin gene that can increase in expression during a compatible plant-pathogen interaction. Received: 15 March 1999 / Accepted: 1 May 1999     

Biosynthesis of ribosome-inactivating proteins from callus and cell suspension cultures of Mirabilis expansa (Ruiz &Pavon)

 Callus and cell suspension cultures from the little known Andean crop Mirabilis expansa were developed and maintained on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/l) and kinetin (0.1 mg/l). Callus and cell suspension cultures were screened with antibodies raised against ME1 (27.5 kDa) and ME2 (27 kDa), two ribosome-inactivating proteins (RIPs) previously found in roots of M. expansa. A 29-kDa protein found in callus and cell suspensions reacted strongly with ME1 antibodies. The 29-kDa protein, named MEC, was purified to homogeneity by ammonium sulfate precipitation and cation exchange perfusion chromatography. Amino acid N-terminal sequencing revealed close homology between MEC and ME1. The MEC amino acid sequence examined was highly conserved among RIPs from widely different sources. This new RIP was immunolocalized to the cell walls of callus and cell suspension cultures. Received: 20 August 1999 / Revision received: 10 December 1999 / Accepted: 21 December 1999     

Proteomic analysis of pathogen-responsive proteins from rice leaves induced by rice blast fungus, Magnaporthe grisea

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

BTH-Induced Accumulation of Extracellular Proteins and Blackspot Disease in Rose

Treatment of rose shoots with 50 µM acibenzolar-S-methyl (BTH) resulted in increased protection against Diplocarpon rosae. This was accompanied by the induction and accumulation of a set of extracellular proteins as shown by SDS-PAGE and 2D-PAGE. Some of these proteins have been identified as PR-1, PR-2, PR-3 and PR-5 proteins by immunoblot analysis probed with tobacco antisera against PR-1c, PR-N, PR-Q and PR-S protein. Most of the extracellular proteins activated by BTH were also induced and found to accumulate in leaves upon infection with Diplocarpon rosae. However, their accumulation was much more pronounced in BTH-pretreated leaves than in water-pretreated leaves upon a challenge inoculation with D. rosae, particularly, the 15 kD PR-1, 36 and 37 kD PR-2 proteins. They may be more important in the expression of disease resistance.