Plant Cell Wall Proteins Research Articles

Overexpression of two cambium‐abundant Chinese fir (Cunninghamia lanceolata) α‐expansin genes ClEXPA1 and ClEXPA2 affect growth and development in transgenic tobacco and increase the amount of cellulose in stem cell walls

Expansins are unique plant cell wall proteins that possess the ability to induce immediately cell wall extension in vitro and cell expansion in vivo. To investigate the biological functions of expansins that are abundant in wood-forming tissues, we cloned two expansin genes from the differentiating xylem of Chinese fir (Cunninghamia lanceolata (Lamb.) Hook). Phylogenetic reconstruction indicated that they belong to α-expansin (EXPA), named ClEXPA1 and ClEXPA2. Expression pattern analysis demonstrated that they are preferentially expressed in the cambium region. Overexpression of ClEXPA1 and ClEXPA2 in tobacco plants yielded pleiotropic phenotypes of plant height, stem diameter, leaf number and seed pod. The height and diameter growth of the 35S(pro) :ClEXPA1 and 35S(pro) :ClEXPA2 transgenic plants were increased drastically, exhibiting an enlargement of pith parenchyma cell size. Isolated cell walls of ClEXPA1 and ClEXPA2 overexpressors contained 30%-50% higher cellulose contents than the wild type, accompanied by a thickening of the cell walls in the xylem region. Both ClEXPA1 and ClEXPA2 are involved in plant growth and development, with a partially functional overlap. Expansins are not only able to induce cell expansion in different tissues/organs in vivo, but they also can act as a potential activator during secondary wall formation by directly or indirectly affecting cellulose metabolism, probably in a cell type-dependent manner.

A bioinformatics approach to the identification, classification, and analysis of hydroxyproline-rich glycoproteins.

Hydroxyproline-rich glycoproteins (HRGPs) are a superfamily of plant cell wall proteins that function in diverse aspects of plant growth and development. This superfamily consists of three members: hyperglycosylated arabinogalactan proteins (AGPs), moderately glycosylated extensins (EXTs), and lightly glycosylated proline-rich proteins (PRPs). Hybrid and chimeric versions of HRGP molecules also exist. In order to "mine" genomic databases for HRGPs and to facilitate and guide research in the field, the BIO OHIO software program was developed that identifies and classifies AGPs, EXTs, PRPs, hybrid HRGPs, and chimeric HRGPs from proteins predicted from DNA sequence data. This bioinformatics program is based on searching for biased amino acid compositions and for particular protein motifs associated with known HRGPs. HRGPs identified by the program are subsequently analyzed to elucidate the following: (1) repeating amino acid sequences, (2) signal peptide and glycosylphosphatidylinositol lipid anchor addition sequences, (3) similar HRGPs via Basic Local Alignment Search Tool, (4) expression patterns of their genes, (5) other HRGPs, glycosyl transferase, prolyl 4-hydroxylase, and peroxidase genes coexpressed with their genes, and (6) gene structure and whether genetic mutants exist in their genes. The program was used to identify and classify 166 HRGPs from Arabidopsis (Arabidopsis thaliana) as follows: 85 AGPs (including classical AGPs, lysine-rich AGPs, arabinogalactan peptides, fasciclin-like AGPs, plastocyanin AGPs, and other chimeric AGPs), 59 EXTs (including SP(5) EXTs, SP(5)/SP(4) EXTs, SP(4) EXTs, SP(4)/SP(3) EXTs, a SP(3) EXT, "short" EXTs, leucine-rich repeat-EXTs, proline-rich extensin-like receptor kinases, and other chimeric EXTs), 18 PRPs (including PRPs and chimeric PRPs), and AGP/EXT hybrid HRGPs.

Promotion of Efficient Saccharification of Crystalline Cellulose by Aspergillus fumigatus Swo1

Swollenin is a protein from Trichoderma reesei that has a unique activity for disrupting cellulosic materials, and it has sequence similarity to expansins, plant cell wall proteins that have a loosening effect that leads to cell wall enlargement. In this study we cloned a gene encoding a swollenin-like protein, Swo1, from the filamentous fungus Aspergillus fumigatus, and designated the gene Afswo1. AfSwo1 has a bimodular structure composed of a carbohydrate-binding module family 1 (CBM1) domain and a plant expansin-like domain. AfSwo1 was produced using Aspergillus oryzae for heterologous expression and was easily isolated by cellulose-affinity chromatography. AfSwo1 exhibited weak endoglucanase activity toward carboxymethyl cellulose (CMC) and bound not only to crystalline cellulose Avicel but also to chitin, while showing no detectable affinity to xylan. Treatment by AfSwo1 caused disruption of Avicel into smaller particles without any detectable reducing sugar. Furthermore, simultaneous incubation of AfSwo1 with a cellulase mixture facilitated saccharification of Avicel. Our results provide a novel approach for efficient bioconversion of crystalline cellulose into glucose by use of the cellulose-disrupting protein AfSwo1.

Nonaqueous titration of amino groups in polymeric matrix of plant cell walls

Nonaqueous titration was used for detection of free amino groups in the polymeric matrix of plant cell walls. The content of amino groups varied in the range 0.54-0.91 and total nitrogen in the range 1.0-4.2 mmol per gram dry mass of cell walls depending on the plant species. However, these data on the high content of free amino groups do not correlate with the present day concept that the nitrogen fraction in charged amino groups in plant cell wall proteins, which are assumed to be mainly amino groups of lysine and arginine residues, is about 10%. It is supposed that most detected free amino groups belong to the hydroxy-amino acids hydroxyproline and tyrosine that can be bound at the hydroxyl group with the carbohydrate part of glycoprotein or another structural cell wall polymer.

A simplified management of the in situ evaluation of feedstuffs in ruminants: Application to the study of the digestive availability of protein and amino acids corrected for the ruminal microbial contamination

The ruminal effective degradability (RED) and intestinal effective digestibility (IED) for dry matter, crude protein (CP) and amino acids (AA) were estimated by a simplified in situ method using pooled samples from rumen-incubated residues, which represented the ruminal outflow of undegraded feed. The effect of microbial contamination in the rumen was corrected using 15N infusion techniques. Studies were carried out for soybean meal (SBM), barley grain (BG) and lucerne hay (LH) in three wethers cannulated in the rumen and the duodenum. Uncorrected values of RED for CP obtained either by mathematical integration or our simplified method were similar in all feeds. Microbial N in the pooled samples of SBM, BG and LH were 2%, 11% and 24% of total N, respectively. However, intestinal incubation eliminated this microbial charge by 100%, 99% and 88%, respectively. With microbial corrections, RED showed an increase, and IED showed a decrease, except for SBM. With this correction, intestinal digested CP was reduced by 2% in SBM, 13% in BG and 34% in LH. Corrected IED of AA was relatively similar in SBM (97–99%). However, large variations were observed in BG (74–93%) and in LH (10–88%). Digestion in the rumen and intestine changed the essential AA pattern. Overall, our results support that AA digestion is affected by the characteristics of their radicals and their contents in plant cell wall proteins. The accurate estimation of feed metabolisable AA or protein requires effective measures that are corrected by ruminal microbial contamination. The proposed in situ method largely simplifies these tasks and allows a more complete and less expensive feed evaluation.

Three aspartic acid residues of polygalacturonase-inhibiting protein (PGIP) fromPhaseolus vulgarisare critical for inhibition ofFusarium phyllophilumPG

Polygalacturonase-inhibiting proteins (PGIPs) are plant cell wall proteins that specifically inhibit the activity of endopolygalacturonases (PGs) produced by fungi during the infection process. The interaction with PGIPs limits the destructive potential of PGs and may trigger plant defence responses through the release of elicitor active oligogalacturonides. In order to pinpoint the residues of PvPGIP2 from Phaseolus vulgaris involved in the interaction with PGs, we used site-directed mutagenesis to mutate the residues D131, D157 and D203, and tested for the inhibitory activity of the mutant proteins expressed in Pichia pastoris against Fusarium phyllophilum and Aspergillus niger PGs. Here, we report that mutation of these residues affects the inhibition capacity of PvPGIP2 against F. phyllophilum PG.

EglD, a putative endoglucanase, with an expansin like domain is localized in the conidial cell wall of Aspergillus nidulans

Although the process of conidial germination in filamentous fungi has been extensively studied, many aspects remain to be elucidated since the asexual spore or conidium is vital in their life cycle. Breakage and reformation of cell wall polymer bonds along with the maintenance of cell wall plasticity during conidia germination depend upon a range of hydrolytic enzymes whose activity is analogous to that of expansins, a highly conserved group of plant cell wall proteins with characteristic wall loosening activity. In the current study, we identified and characterized the eglD gene in Aspergillus nidulans, an expansin-like gene the product of which shows strong similarities with bacterial and fungal endo-β1,4-glucanases. However, we failed to show such activity in vitro. The eglD gene is constitutively expressed in all developmental stages and compartments of A. nidulans asexual life cycle. However, the EglD protein is exclusively present in conidial cell walls. The role of the EglD protein in morphogenesis, growth and germination rate of conidia was investigated. Our results show that EglD is a conidial cell wall localized expansin-like protein, which could be involved in cell wall remodeling during germination.

Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.

Activity of endo-polygalacturonases in mirid bugs (Heteroptera: Miridae) and their inhibition by plant cell wall proteins (PGIPs)

Endo-polygalacturonases (PGs) are hydrolytic enzymes involved in the degradation of pectin, one of the major components of plant cell wall. While PGs from fungi, bacteria and plants have been extensively studied, PGs from insects are much less known, although they are likely to play an important role in insect-plant interactions. Presence of PGs has been reported for both piercing-sucking and chewing insect species, and possibly more commonly in mirid bugs (Heteroptera: Miridae). A screening of some common mirid species and other insects, belonging to different orders and families, was conducted using agarose diffusion assays run at different pHs. All the mirid species tested [Lygus rugulipennis Popp., L. pratensis (L.), Orthops kalmi (L.), Adelphocoris lineolatus (Goeze) and Closterotomus norwegicus (Gmelin)] showed PG activity, mainly at pH 7-8, whereas no activity was recorded for the other insect species, except Sitophilus sp. (Coleoptera: Curculionidae). PG activity in females of L. pratensis was significantly higher than in males, whereas there were no differences between the sexes in the other species. In all these species, PGs were present both in the salivary glands and the gut, with a higher activity in the salivary glands, confirming the role of these enzymes in the feeding behaviour of mirid bugs. Inhibition of mirid PGs by polygalacturonase-inhibiting proteins (PGIPs) from different plant sources was analysed at pH 7. PGIPs are extracellular plant proteins known for their ability to inhibit fungal PGs and restrict fungal colonization. Two PGIPs from Phaseolus vulgaris (PvPGIP3 and PvPGIP4) inhibited PGs of all the mirid bugs tested. This information may be helpful for the development of innovative insect-resistant plant varieties, for use in low-impact IPM.

Nitric oxide functions in the plant hypersensitive disease resistance response

Nitric oxide (NO) is a highly reactive molecule that rapidly diffuses and permeates cell membranes. In animals, NO is implicated in a number of diverse physiological processes such as neurotransmission, vascular smooth muscle relaxation, and platelet inhibition. It may have beneficial effects, for example as a messenger in immune responses, but it is also potentially toxic when the antioxidant system is weak and an excess of reactive oxygen species (ROS) accumulates. During the last few years NO as been detected also in several plant species, and the increasing number of reports on its function in plants have implicated NO as an important effector of growth, development, and defence [1]. The innate immune system of organisms as diverse as vertebrates, invertebrates, and plants shows several characteristics similar with respect to involvement of NO. In the mammalian immune system, NO cooperates with ROS to induce apoptosis of tumor cells and macrophage killing of bacteria. In plants a similar mechanism have evolved to prevent tissue invasion by pathogens. Attempted infection by an avirulent pathogen elicits a battery of defences often accompanied by collapse of challenged host cells thus delimiting the infected zone and avoiding the multiplication and spread of the pathogen. The rapid accumulation of ROS and NO through the activation of enzyme systems similar to neutrophil NADPH oxidase and nitric oxide synthase is one of the earliest events in this hypersensitive reaction (HR). Several lines of evidence suggest that death of host cells during the HR results from the activation of a cell death program, encoded by the plant genome and activated by a fine modulation of O2 -, H2O2 and NO levels [2]. Both NO and ROS are necessary to trigger host cell death (see Figure ​Figure1);1); they are also components of a highly amplified and integrated defence system that involves activation of ion fluxes, changes in protein phosphorylation patterns, extracellular pH, membrane potential, oxidative cross-linking of plant cell wall proteins, and perturbations in the level of cytosolic Ca2+ that triggers the local expression of resistance genes [3]. Figure 1 NO and H2O2 mediated plant cell death. NO, nitric oxide; NOS, nitric oxide synthase; ONOO-, peroxynitrite; H2O2, hydrogen peroxide; GPX, glutathione peroxidase; GST, glutathione S-transferase; SOD, superoxide dismutase; MAPK, mitogen-activated protein ... NO also functions independently of ROS in the induction of various defence genes including pathogenesis-related proteins and enzymes of phenylpropanoid metabolism involved in the production of lignin, antibiotics and the secondary signal salicylic acid (see Figure ​Figure2).2). The mobile nature of NO and its chemical reactivity with various cellular targets means that downstream effects of NO may be directly induced by interaction with various cellular components, like ion channels or proteins that modulates gene expression, or indirectly following interaction with signalling proteins such as protein kinases. NO signalling functions depend on its reactivity and ROS are key modulators of NO in triggering cell death, although through mechanisms different from those commonly observed in animals [4]. Figure 2 Representation of NO signalling functions during the HR. CHS, chalcone synthase; CA, cinnamic acid; Ca2+, calcium influx; cADPR, cyclic cADP ribose; cGMP, cyclic GMP; MAPK, mitogen-activated protein kinase; PAL, phenylalanine ammonia lyase; PHE, phenylalanine; ... In summary, although several hypotheses still await experimental demonstration, it is now clear that NO is an important component of plant defence systems. Much evidence supports the view that NO plays a key role in disease resistance responses. Moreover, the recent identification of a plant NOS will lead to the characterization and manipulation of mechanisms modulating NO signalling. Thus, the understanding of NO signalling functions at the biochemical, cellular and molecular levels will soon make it possible to discern several important physiological and pathological processes in plants, as has already been demonstrated in mammals.

Evaluation of tolerance to Pierce's disease and Botrytis in transgenic plants of Vitis vinifera L. expressing the pear PGIP gene

SUMMARY Polygalacturonase-inhibiting proteins (PGIPs) are plant cell-wall proteins that specifically inhibit fungal endo-polygalacturonases (PGs) that contribute to the aggressive decomposition of susceptible plant tissues. The inhibition of fungal PGs by PGIPs suggests that PGIPs have a role in plant tolerance to fungal infections and this has been observed in transgenic plants expressing PGIPs. Xylella fastidiosa, the causal agent of Pierce's disease (PD) in grapevines, has genes that encode cell-wall-degrading enzymes, including a putative PG. Therefore, we hypothesized that PGIP expression could confer tolerance against this bacterium as well as against the fungal pathogen Botrytis cinerea. To test this hypothesis, Vitis vinifera cvs. 'Thompson Seedless' and 'Chardonnay' were transformed to express pear fruit PGIP-encoding gene (pPGIP) under the control of the CaMV 35S promoter. Substantial pear PGIP (pPGIP) activity was found in crude extracts from leaves and in xylem exudate of transgenic lines obtained from independent transformation events, but not in untransformed controls. pPGIP activity was detected in xylem exudate of untransformed scions grafted on to transgenic rootstocks expressing pPGIP. Leaves of transgenic plants infected with B. cinerea had reduced rates of lesion expansion. The development of PD was delayed in some transgenic lines with increased pPGIP activity. PD-tolerant transgenic lines had reduced leaf scorching, lower Xylella titres and better re-growth after pruning than the untransformed controls.

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3–4 times more enriched in salt stressed epicotyls. SDS–PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.

EVALUATION OF TOLERANCE TO PIERCE'S DISEASE AND BOTRYTIS IN TRANSGENIC PLANTS OF VITIS VINIFERA L. EXPRESSING THE PEAR PGIP GENE

PG-inhibiting proteins (PGIPs) are leucine rich repeat plant cell wall proteins that specifically inhibit fungal polygalacturonases (PGs). Their role in plant defense response suggests that they may be useful for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection. In addition, the fact that Xylella fastidiosa, the causal agent of Pierce’s Disease (PD) in grapevines, has genes putatively encoding PG and other cell wall-degrading enzymes led us to the hypothesis that PGIP could confer resistance against this bacterium. In order to test this hypothesis, proembryogenic calli originating from anthers of Vitis vinifera cvs. Thompson Seedless and Chardonnay were co-cultivated with Agrobacterium tumefaciens strain EHA 101 harboring binary plasmid pDU94.0928 that contains the pear pgip gene under the control of the CaMV 35S promoter. Plants from 51 independently isolated lines have been transferred to the greenhouse. Putative transgenic lines growing in the greenhouse were analyzed by PCR, Western blotting and PGIP activity assays. Western blot analysis demonstrated the presence of the protein in roots, leaves and young stems of the transgenic plants but not in untransformed controls. High levels of enzyme activity have been found in crude extracts from leaves of transgenic lines obtained from independent transformation events but not in untransformed controls. Preliminary results have shown that the development of PD in the transgenic lines analyzed so far is delayed. We also present results regarding the disease response of greenhouse-grown plants after inoculation with Botrytis cinerea.

Cloning of a pine germin-like protein (GLP) gene promoter and analysis of its activity in transgenic tobacco Bright Yellow 2 cells.

Germins and germin-like proteins (GLPs) constitute a large and highly diverse family of ubiquitous plant cell wall proteins. These proteins seem to be involved in many developmental stages and stress-related processes, but their exact participation in these processes generally remains obscure. In Pinus caribaea Morelet, the PcGER1 gene is expressed uniquely in embryo tissues, and encodes a GLP ionically bound to the walls of pine embryo cells maintained in 2,4-D-containing medium. We have cloned a genomic fragment including the 1520 bp 5'-upstream promoter region of PcGER1. This sequence contains, in its 1200 bp distal part, several cis elements (e.g. SEF4, 60 kDa protein, ABA RE and Dof recognition sites) present in genes responding to hormones and/or expressed in embryo or seed tissues, or during germination. The PcGER1 promoter sequence was cloned upstream of the GUS (beta-glucuronidase) reporter gene and transferred to tobacco Bright Yellow 2 (BY-2) cells via Agrobacterium tumefaciens-mediated transformation. Promoter activity and growth performances of transgenic asynchronous cell suspensions were analysed in the presence or absence of 2,4-D and/or BA. Optimal growth, maximum cell-wall yield and PcGER1 promoter activity were observed in the presence of 2,4-D and BA at day 4, the end of the exponential growth phase where 70-75% cells have a 2C DNA content. Analysis of promoter activity during the cell cycle in an aphidicoline-synchronized culture suggested that the expression is maximum in G1 cells. We also showed that under optimal growth conditions, 5' promoter deletions decreased the activity of the reporter gene. We discuss the function of this gene with regards to cell growth. Accession number: The PcGER1 promoter sequence was submitted to the genbank database under the accession number AY077704.

Characterization of two closely linked soybean pgip genes and transcript regulation following pathogen infection and wounding

Polygalacturonase-inhibiting protein (PGIP) is a plant cell wall protein that regulates the action of fungal endopolygalacturonases (PG) favouring the formation of oligogalacturonides active as elicitors of plant defense responses. We have isolated two novel soybean PGIP genes (Gmpgip1 and Gmpgip2) and their recognition specificities against fungal PGs have been tested by expressing them in Nicotiana benthamiana using the Potato virus X (PVX) as vector and in Escherichia coli. In both systems GmPGIP1 and GmPGIP2 accumulate but showed no activity against PGs. Transcript regulation of GmPGIP1 and GmPGIP2 showed that these genes undergo a differential regulation following Sclerotinia sclerotiorum infection and wounding.

Swollenin, a Trichoderma reesei protein with sequence similarity to the plant expansins, exhibits disruption activity on cellulosic materials.

Plant cell wall proteins called expansins are thought to disrupt hydrogen bonding between cell wall polysaccharides without hydrolyzing them. We describe here a novel gene with sequence similarity to plant expansins, isolated from the cellulolytic fungus Trichoderma reesei. The protein named swollenin has an N-terminal fungal type cellulose binding domain connected by a linker region to the expansin-like domain. The protein also contains regions similar to mammalian fibronectin type III repeats, found for the first time in a fungal protein. The swollenin gene is regulated in a largely similar manner as the T. reesei cellulase genes. The biological role of SWOI was studied by disrupting the swo1 gene from T. reesei. The disruption had no apparent effect on the growth rate on glucose or on different cellulosic carbon sources. Non-stringent Southern hybridization of Trichoderma genomic DNA with swo1 showed the presence of other swollenin-like genes, which could substitute for the loss of SWOI in the disruptant. The swollenin gene was expressed in yeast and Aspergillus niger var. awamori. Activity assays on cotton fibers and filter paper were performed with concentrated SWOI-containing yeast supernatant that disrupted the structure of the cotton fibers without detectable formation of reducing sugars. It also weakened filter paper as assayed by an extensometer. The SWOI protein was purified from A. niger var. awamori culture supernatant and used in an activity assay with Valonia cell walls. It disrupted the structure of the cell walls without producing detectable amounts of reducing sugars.

Regulation of acp1, encoding a non-aspartyl acid protease expressed during pathogenesis of Sclerotinia sclerotiorum.

When grown in the presence of sunflower cell walls, Sclerotinia sclerotiorum, an ubiquitous necrotrophic fungus, secretes several acid proteases including a non-aspartyl protease. The gene acp1, encoding an acid protease, has been cloned and sequenced. The intronless ORF encodes a preproprotein of 252 aa and a mature protein of 200 residues. In vitro expression of acp1 is subject to several transcriptional regulatory mechanisms. Expression induced by plant cell-wall proteins is controlled by both carbon and nitrogen catabolite repression. Glucose on its own represses acp1 expression while ammonium repression requires the simultaneous presence of a carbon source. Ambient pH higher than pH 5 overrides induction resulting in full repression of acp1. These transcriptional regulatory mechanisms and the presence of several motifs in the promoter of acp1 that may encode binding sites for the regulators CREA, AREA and PacC suggest the involvement of these regulators in the control of acp1 expression. acp1 is expressed in planta during sunflower cotyledon infection. Expression is low at the beginning of infection but increases suddenly at the stage of necrosis spreading. Comparison of in vitro and in planta acp1 expression suggests that glucose and nitrogen starvation together with acidification can be considered as key factors controlling Scl. sclerotiorum gene expression during pathogenesis.

The distribution and expression of a biotrophy‐related gene,CIH1, within the genusColletotrichum

Abstract During the biotrophic phase of the infection process of the hemibiotrophic anthracnose fungus Colletotrichum lindemuthianum, an intracellular hypha develops within epidermal cells of its host, Phaseolus vulgaris. This is followed by the formation of secondary hyphae during the necrotrophic phase. Previous work using a monoclonal antibody, UB25, has identified a glycoprotein that is specific to the interfacial matrix that forms between the wall of the intracellular hypha and the invaginated host plasma membrane. The gene encoding the protein identified by UB25 was cloned by immunoscreening and designated CIH1. The predicted amino acid sequence revealed a proline-rich glycoprotein, and biochemical evidence suggested that it formed a cross-linked structure at the biotrophic interface. Although CIH1 is a fungal gene, its product has several similarities to plant cell wall proteins. In this paper, we have surveyed the distribution and expression of CIH1 within the genus Colletotrichum, encompassing both necrotrophic and hemibiotrophic species. The results show that homologues of the CIH1 gene are present in all the Colletotrichum species tested. Northern blot studies of the time course of the infection process in planta have shown that CIH1 is expressed by both C. lindemuthianum in bean and C. trifolii in alfalfa during the biotrophic phase of fungal development. Immunofluorescence labelling of infected epidermal strips with UB25 revealed that the intracellular hyphae formed by C. destructivum as it infects alfalfa were specifically labelled in a similar way to those formed by C. lindemuthianum in bean. Northern and Western analysis showed that CIH1 was also expressed by C. lindemuthianum in vitro, though not constitutively. Overall, the evidence supports a role for CIH1 in biotrophy within the genus Colletotrichum.

Two bean cell wall proteins more abundant during water deficit are high in proline and interact with a plasma membrane protein

Two antigenically related glycoproteins, called p33 and p36, accumulate in the soluble fraction of the cell wall in response to water deficit in Phaseolus vulgaris. In this report, we show that p33 and p36 are able to adhere to leaf protoplasts, and that they bind to plasma membrane (PM) vesicles in a divalent cation-dependent manner. Data from the partial amino acid sequence of the p33 and p36 proteins indicate that they contain repeats of the decapeptide POVYKPOVEK; therefore, they are related to proline-rich proteins. Binding assays demonstrate that both proteins specifically bind to an 80 kDa PM protein. This binding is competed with a peptide that contains the RGD motif, as well as with fibronectin, which also includes this sequence, suggesting that the 80 kDa PM protein has an integrin-like function whose natural ligands are p33 and p36. This is the first case where a PM ligand for a higher plant cell wall protein has been identified.

An expansin gene expressed in ripening strawberry fruit.

Tissue softening accompanies the ripening of many fruit and initiates the processes of irreversible deterioration. Expansins are plant cell wall proteins proposed to disrupt hydrogen bonds within the cell wall polymer matrix. Expression of specific expansin genes has been observed in tomato (Lycopersicon esculentum) meristems, expanding tissues, and ripening fruit. It has been proposed that a tomato ripening-regulated expansin might contribute to cell wall polymer disassembly and fruit softening by increasing the accessibility of specific cell wall polymers to hydrolase action. To assess whether ripening-regulated expansins are present in all ripening fruit, we examined expansin gene expression in strawberry (Fragaria x ananassa Duch.). Strawberry differs significantly from tomato in that the fruit is derived from receptacle rather than ovary tissue and strawberry is non-climacteric. A full-length cDNA encoding a ripening-regulated expansin, FaExp2, was isolated from strawberry fruit. The deduced amino acid sequence of FaExp2 is most closely related to an expansin expressed in early tomato development and to expansins expressed in apricot fruit rather than the previously identified tomato ripening-regulated expansin, LeExp1. Nearly all previously identified ripening-regulated genes in strawberry are negatively regulated by auxin. Surprisingly, FaExp2 expression was largely unaffected by auxin. Overall, our results suggest that expansins are a common component of ripening and that non-climacteric signals other than auxin may coordinate the onset of ripening in strawberry.