A novel Phytophthora infestans haustorium-specific membrane protein is required for infection of potato

Phytophthora infestans causes late-blight, a devastating and re-emerging disease of potato crops. During the early stages of infection, P. infestans differentiates infection-specific structures such as appressoria for host epidermal cell penetration, followed by infection vesicles, and haustoria to establish a biotrophic phase of interaction. Here we report the cloning, from a suppression subtractive hybridization library, of a P. infestans gene called Pihmp1 encoding a putative glycosylated protein with four closely spaced trans-membrane helices. Pihmp1 expression is upregulated in germinating cysts and in germinating cysts with appressoria, and significantly upregulated throughout infection of potato. Transient gene silencing of Pihmp1 led to loss of pathogenicity and indicated involvement of this gene in the penetration and early infection processes of P. infestans. P. infestans transformants expressing a Pihmp1::monomeric red fluorescent protein (mRFP) fusion demonstrated that Pihmp1 was translated in germinating sporangia, germinating cysts and appressoria, accumulated in the appressorium, and was located at the haustorial membrane during infection. Furthermore, we discovered that haustorial structures are formed over a 3 h period, maturing for up to 12 h, and that their formation is initiated only at sites on the surface of intercellular hyphae where Pihmp1::mRFP is localized. We propose that Pihmp1 is an integral membrane protein that provides physical stability to the plasma membrane of P. infestans infection structures. We have provided the first evidence that the surface of oomycete haustoria possess proteins specific to these biotrophic structures, and that formation of biotrophic structures (infection vesicles and haustoria) is essential to successful host colonization by P. infestans.     

Mandipropamid targets the cellulose synthase-like PiCesA3 to inhibit cell wall biosynthesis in the oomycete plant pathogen, Phytophthora infestans

Oomycete plant pathogens cause a wide variety of economically and environmentally important plant diseases. Mandipropamid (MPD) is a carboxylic acid amide (CAA) effective against downy mildews, such as Plasmopara viticola on grapes and potato late blight caused by Phytophthora infestans . Historically, the identification of the mode of action of oomycete-specific control agents has been problematic. Here, we describe how a combination of biochemical and genetic techniques has been utilized to identify the molecular target of MPD in P. infestans . Phytophthora infestans germinating cysts treated with MPD produced swelling symptoms typical of cell wall synthesis inhibitors, and these effects were reversible after washing with H₂O. Uptake studies with ¹⁴C-labelled MPD showed that this oomycete control agent acts on the cell wall and does not enter the cell. Furthermore, ¹⁴C glucose incorporation into cellulose was perturbed in the presence of MPD which, taken together, suggests that the inhibition of cellulose synthesis is the primary effect of MPD. Laboratory mutants, insensitive to MPD, were raised by ethyl methane sulphonate (EMS) mutagenesis, and gene sequence analysis of cellulose synthase genes in these mutants revealed two point mutations in the PiCesA3 gene, known to be involved in cellulose synthesis. Both mutations in the PiCesA3 gene result in a change to the same amino acid (glycine-1105) in the protein. The transformation and expression of a mutated PiCesA3 allele was carried out in a sensitive wild-type isolate to demonstrate that the mutations in PiCesA3 were responsible for the MPD insensitivity phenotype.     

Knockout of the AtCESA2 gene affects microtubule orientation and causes abnormal cell expansion in Arabidopsis

Complete cellulose synthesis is required to form functional cell walls and to facilitate proper cell expansion during plant growth. AtCESA2 is a member of the cellulose synthase A family in Arabidopsis (Arabidopsis thaliana) that participates in cell wall formation. By analysis of transgenic seedlings, we demonstrated that AtCESA2 was expressed in all organs, except root hairs. The atcesa2 mutant was devoid of AtCESA2 expression, leading to the stunted growth of hypocotyls in seedlings and greatly reduced seed production in mature plants. These observations were attributed to alterations in cell size as a result of reduced cellulose synthesis in the mutant. The orientation of microtubules was also altered in the atcesa2 mutant, which was clearly observed in hypocotyls and petioles. Complementary expression of AtCESA2 in atcesa2 could rescue the mutant phenotypes. Together, we conclude that disruption of cellulose synthesis results in altered orientation of microtubules and eventually leads to abnormal plant growth. We also demonstrated that the zinc finger-like domain of AtCESA2 could homodimerize, possibly contributing to rosette assemblies of cellulose synthase A within plasma membranes.     

Cyst germination proteins of the potato pathogen Phytophthora infestans share homology with human mucins

We have cloned genes of Phytophthora infestans, the causal agent of potato late blight, that are activated shortly before the onset of invasion of the host tissue. The three genes isolated appear to be arranged in a genomic cluster and belong to a small polymorphic gene family. A conspicuous feature of the deduced proteins is an internal octapeptide repeat with the consensus sequence TTYAP TEE. Because of this structural motif, these novel P. infestans proteins were named Car (Cyst-germination-specific acidic repeat) proteins. One of the genes, car90, codes for 1,489 amino acids including 120 octapeptide tandem repeats. Car proteins are transiently expressed during germination of cysts and formation of appressoria and are localized at the surface of germlings. The structural motif of tandemly repeated oligopeptides also occurs in a prominent class of proteins, the mucins, from mammals. The P. infestans Car proteins share 51% sequence homology with the tandem repeat region of a subfamily of human mucins. According to the physiological functions ascribed to mucins, we suggest that Car proteins may serve as a mucous cover protecting the germling from desiccation, physical damage, and adverse effects of the plant defense response and may assist in adhesion to the leaf surface.     

Three AtCesA6‐like members enhance biomass production by distinctively promoting cell growth in Arabidopsis

Cellulose is an abundant biopolymer and a prominent constituent of plant cell walls. Cellulose is also a central component to plant morphogenesis and contributes the bulk of a plant's biomass. While cellulose synthase (CesA) genes were identified over two decades ago, genetic manipulation of this family to enhance cellulose production has remained difficult. In this study, we show that increasing the expression levels of the three primary cell wall AtCesA6‐like genes (AtCesA2, AtCesA5, AtCesA6), but not AtCesA3, AtCesA9 or secondary cell wall AtCesA7, can promote the expression of major primary wall CesA genes to accelerate primary wall CesA complex (cellulose synthase complexes, CSCs) particle movement for acquiring long microfibrils and consequently increasing cellulose production in Arabidopsis transgenic lines, as compared with wild‐type. The overexpression transgenic lines displayed changes in expression of genes related to cell growth and proliferation, perhaps explaining the enhanced growth of the transgenic seedlings. Notably, overexpression of the three AtCesA6‐like genes also enhanced secondary cell wall deposition that led to improved mechanical strength and higher biomass production in transgenic mature plants. Hence, we propose that overexpression of certain AtCesA genes can provide a biotechnological approach to increase cellulose synthesis and biomass accumulation in transgenic plants.     

A Kazal-like extracellular serine protease inhibitor from Phytophthora infestans targets the tomato pathogenesis-related protease P69B

The oomycetes form one of several lineages within the eukaryotes that independently evolved a parasitic lifestyle and consequently are thought to have developed alternative mechanisms of pathogenicity. The oomycete Phytophthora infestans causes late blight, a ravaging disease of potato and tomato. Little is known about processes associated with P. infestans pathogenesis, particularly the suppression of host defense responses. We describe and functionally characterize an extracellular protease inhibitor, EPI1, from P. infestans. EPI1 contains two domains with significant similarity to the Kazal family of serine protease inhibitors. Database searches suggested that Kazal-like proteins are mainly restricted to animals and apicomplexan parasites but appear to be widespread and diverse in the oomycetes. Recombinant EPI1 specifically inhibited subtilisin A among major serine proteases and inhibited and interacted with the pathogenesis-related P69B subtilisin-like serine protease of tomato in intercellular fluids. The epi1 and P69B genes were coordinately expressed and up-regulated during infection of tomato by P. infestans. Inhibition of tomato proteases by EPI1 could form a novel type of defense-counterdefense mechanism between plants and microbial pathogens. In addition, this study points to a common virulence strategy between the oomycete plant pathogen P. infestans and several mammalian parasites, such as the apicomplexan Toxoplasma gondii.     

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.     

Patterns of diversifying selection in the phytotoxin-like scr74 gene family of Phytophthora infestans

Phytophthora infestans, the organism responsible for the Irish famine, causes late blight, a re-emerging disease of potato and tomato. Little is known about the molecular evolution of P. infestans genes. To identify candidate effector genes (virulence or avirulence genes) that may have co-evolved with the host, we mined expressed sequence tag (EST) data from infection stages of P. infestans for secreted and potentially polymorphic genes. This led to the identification of scr74, a gene that encodes a predicted 74-amino acid secreted cysteine-rich protein with similarity to the Phytophthora cactorum phytotoxin PcF. The expression of scr74 was upregulated approximately 60-fold 2 to 4 days after inoculation of tomato and was also significantly induced during early stages of colonization of potato. The scr74 gene was found to belong to a highly polymorphic gene family within P. infestans with 21 different sequences identified. Using the approximate and maximum likelihood (ML) methods, we found that diversifying selection likely caused the extensive polymorphism observed within the scr74 gene family. Pairwise comparisons of 17 scr74 sequences revealed elevated ratios of nonsynonymous to synonymous nucleotide-substitution rates, particularly in the mature region of the proteins. Using ML, all 21 polymorphic amino acid sites were identified to be under diversifying selection. Of these 21 amino acids, 19 are located in the mature protein region, suggesting that selection may have acted on the functional portions of the proteins. Further investigation of gene copy number and organization revealed that the scr74 gene family comprises at least three copies located in a region of no more than 300 kb of the P. infestans genome. We found evidence that recombination contributed to sequence divergence within at least one gene locus. These results led us to propose an evolutionary model that involves gene duplication and recombination, followed by functional divergence of scr74 genes. This study provides support for using diversifying selection as a criterion for identifying candidate effector genes from sequence databases.     

Arabidopsis fragile fiber8, which encodes a putative glucuronyltransferase, is essential for normal secondary wall synthesis

Secondary walls in vessels and fibers of dicotyledonous plants are mainly composed of cellulose, xylan, and lignin. Although genes involved in biosynthesis of cellulose and lignin have been intensively studied, little is known about genes participating in xylan synthesis. We found that Arabidopsis thaliana fragile fiber8 (fra8) is defective in xylan synthesis. The fra8 mutation caused a dramatic reduction in fiber wall thickness and a decrease in stem strength. FRA8 was found to encode a member of glycosyltransferase family 47 and exhibits high sequence similarity to tobacco (Nicotiana plumbaginifolia) pectin glucuronyltransferase. FRA8 is expressed specifically in developing vessels and fiber cells, and FRA8 is targeted to Golgi. Comparative analyses of cell wall polysaccharide fractions from fra8 and wild-type stems showed that the xylan and cellulose contents are drastically reduced in fra8, whereas xyloglucan and pectin are elevated. Further structural analysis of cell walls revealed that although wild-type xylans contain both glucuronic acid and 4-O-methylglucuronic acid residues, xylans from fra8 retain only 4-O-methylglucuronic acid, indicating that the fra8 mutation results in a specific defect in the addition of glucuronic acid residues onto xylans. These findings suggest that FRA8 is a glucuronyltransferase involved in the biosynthesis of glucuronoxylan during secondary wall formation.     

The histopathology of Phaeocryptopus gaeumannii on Douglas-fir needles

Germinating ascospores of Phaeocryptopus gaeumannii produce suprastomatal appressoria from which penetration pegs enter needles. Initial infection occurs between late May and early Jul and coincides with budbreak and shoot elongation. Colonization within needles is exclusively intercellular and increases continuously during Jul-May. No intracellular hyphae or haustoria were observed, but hyphae closely appressed to mesophyll and palisade cell walls are abundant by 3-5 mo after initial infection. Pseudothecial primordia begin to form in epistomatal chambers Oct-Apr, 4-9 mo after initial infection. Pseudothecial primordia developing in the epistomatal chamber are connected to the endophytic thallus by specialized cells in the substomatal chamber that have thickened apical walls and resemble phialides but are not involved in asexual reproduction. The apical wall thickenings instead appear to function as reinforcement against the turgor pressure of the guard cells, allowing cytoplasmic continuity to be maintained between the developing pseudothecium and vegetative hyphae within the needle. Concurrent with the formation of pseudothecial primorida, epiphytic hyphae emerge from the periphery of developing pseudothecia, grow across the needle surface, form numerous anastomoses and reenter the needle by producing appressoria above unoccupied stomata. Epiphytic hyphae and their associated appressoria gradually become more abundant during Oct-Jan.     

Direct observation of the structure of mycelial cell walls from the potato blight fungus Phytophthora infestans by solid-state 13C NMR

Mycelial cell walls from the potato blight fungus ( Phytophthora infestans ) (Mont.) de Bary were examined in the solid state by ¹³C nuclear magnetic resonance spectroscopy with cross-polarization and magic-angle spinning. The spectrum was free from interference by spinning sidebands. The main component of the cell walls had the spectral properties of a β -(1,3')-glucan. Protein appeared to be present also. The presence of β -(1,6')-linked glucose residues, cellulose or chitin was not ruled out, but there was no evidence for these as major components of the cell walls.     

Disruption of cellulose synthesis by isoxaben causes tip swelling and disorganizes cortical microtubules in elongating conifer pollen tubes

Summary.  In elongating pollen tubes of the conifer Picea abies (Norway spruce), microtubules form a radial array beneath the plasma membrane only at the elongating tip and an array parallel with elongation throughout the tube. Tips specifically swell following microtubule disruption. Here we test whether these radial microtubules coordinate cell wall deposition and maintain tip integrity as tubes elongate. Control pollen tubes contain cellulose throughout the walls, including the tip. Pollen tubes grown in the presence of isoxaben, which disrupts cellulose synthesis, are significantly shorter with a decrease in cellulose throughout the walls. Isoxaben also significantly increases the frequency of tip swelling, with no effect on tube width outside of the swollen tip. The decrease in cellulose is more pronounced in pollen tubes with swollen tips. The effects of isoxaben are reversible. Following isoxaben treatment, the radial array of microtubules persists beneath the plasma membrane of nonswollen tips, while this array is specifically disrupted in swollen tips. Microtubules instead form a random network throughout the tip. Growth in these pollen tubes is turgor driven, but the morphological changes due to isoxaben are not just the result of weakened cell walls since pollen tubes grown in hypoosmotic media are not significantly shorter but do have swollen tips and tubes are wider along their entire length. We conclude that the radial microtubules in the tip do maintain tip integrity and that the specific inhibition of cellulose microfibril deposition leads to the disorganization of these microtubules. This supports the emerging model that there is bidirectional communication across the plasma membrane between cortical microtubules and cellulose microfibrils. Received January 15, 2002; accepted August 3, 2002; published online March 11, 2003     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.     

Downregulation of the Petunia hybrida alpha-expansin gene PhEXP1 reduces the amount of crystalline cellulose in cell walls and leads to phenotypic changes in petal limbs

The expansins comprise a family of proteins that appear to be involved in the disruption of the noncovalent bonds between cellulose microfibrils and cross-linking glycans, thereby promoting wall creep. To understand better the expansion process in Petunia hybrida (petunia) flowers, we isolated a cDNA corresponding to the PhEXP1 alpha-expansin gene of P. hybrida. Evaluation of the tissue specificity and temporal expression pattern demonstrated that PhEXP1 is preferentially expressed in petal limbs during development. To determine the function of PhEXP1, we used a transgenic antisense approach, which was found to cause a decrease in petal limb size, a reduction in the epidermal cell area, and alterations in cell wall morphology and composition. The diminished cell wall thickness accompanied by a reduction in crystalline cellulose indicates that the activity of PhEXP1 is associated with cellulose metabolism. Our results suggest that expansins play a role in the assembly of the cell wall by affecting either cellulose synthesis or deposition.     

Inhibitory effect of a defensin gene from the Andean crop maca (Lepidium meyenii) against Phytophthora infestans

In this study, we report the isolation of a defensin gene, lm-def, isolated from the Andean crop 'maca' (Lepidium meyenii) with activity against the pathogen Phytophthora infestans responsible of late blight disease of the potato and tomato crops. The lm-def gene has been isolated by polymerase chain reaction (PCR) using degenerate primers corresponding to conserved regions of 13 plant defensin genes of the Brassicaceae family assuming that defensin genes are highly conserved among cruciferous species. The lm-def gene belongs to a small multigene family of at least 10 members possibly including pseudogenes as assessed by genomic hybridization and nucleotide sequence analyses. The deduced mature Lm-Def peptide is 51 amino acids in length and has 74-94% sequence identity with other plant defensins of the Brassicaceae family. The Lm-Def peptide was produced as a fusion protein using the pET-44a expression vector and purified using an immobilized metal ion affinity chromatography. The recombinant protein (NusA:Lm-Def) exhibited in vitro activity against P. infestans. The NusA:Lm-Def protein caused growth inhibition and hyphal damage at concentration not greater than 0.4 microM. In contrast, the NusA protein alone expressed and purified similarly did not show any activity against P. infestans. Therefore, these results indicate that the lm-def gene isolated from maca belong to the plant defensin family with activity against P. infestans. Its expression in potato, as a transgene, might help to control the late blight disease caused by P. infestans with the advantage of being of plant origin.