Reversible cytoplasmic rearrangements precede wall apposition,hypersensitive cell death and defense-related gene activation in potato/Phytophthora infestans interactions

We used video microscopy techniques as a tool for live examination of the dynamic aspects of plant/fungus interactions. Early, dynamic responses of epidermal midrib cells of leaves from a potato cultivar (Solanum tuberosum L. cv. Datura) carrying resistance gene R1 to Phytophthora infestans (race 1: compatible interaction, race 4: incompatible interaction) were monitored. Similar responses were observed in both types of interaction, ranging from no visible reaction of invaded plant cells to hypersensitive cell death. The overall defense response of each individual cell exhibited a highly dynamic behavior that appeared to be tightly coordinated with the growth of the fungus. Initial localized reactions, including major rearrangements within the cytoplasm, occurred directly at the fungal penetration site, where rapid apposition of autofluorescent material and callose took place. If fungal invasion stopped at this stage, the host cell restored its normal cytoplasmic activity and survived. Hypersensitive cell death occurred only when fungal growth had proceeded to the formation of a clearly identifiable haustorium. In such cases, cytoplasm and nucleus conglomerated around the intracellular fungal structure, followed by a sudden collapse of the whole conglomerate and an instantaneous collapse of the fungal haustorium. Only small quantitative differences between the compatible and incompatible interactions of the two fungal races were observed for these early responses of epidermal cells. In the incompatible interaction, a slightly larger number of epidermal cells responded to fungal attack. More pronounced quantitative differences between compatible and incompatible interactions occurred upon fungal invasion of the mesophyll. These differences in the number of responding cells were not reflected at the level of gene expression: the spatial and temporal activation patterns of two defense-related genes, encoding phenylalanine ammonia-lyase and pathogenesis-related protein 1, were similar in both types of interaction.Dedicated to Professor Peter Sitte, Freiburg, Germany, on the occasion of his 65th birthday     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Establishment of compatibility in the Ustilago maydis/maize pathosystem

The fungus Ustilago maydis is a biotrophic pathogen parasitizing on maize. The most prominent symptoms of the disease are large tumors in which fungal proliferation and spore differentiation occur. In this study, we have analyzed early and late tumor stages by confocal microscopy. We show that fungal differentiation occurs both within plant cells as well as in cavities where huge aggregates of fungal mycelium develop. U. maydis is poorly equipped with plant CWDEs and we demonstrate by array analysis that the respective genes follow distinct expression profiles at early and late stages of tumor development. For the set of three genes coding for pectinolytic enzymes, deletion mutants were generated by gene replacement. Neither single nor triple mutants were affected in pathogenic development. Based on our studies, we consider it unlikely that U. maydis feeds on carbohydrates derived from the digestion of plant cell wall material, but uses its set of plant CWDEs for softening the cell wall structure as a prerequisite for in planta growth.     

Role of calcium in signal transduction during the hypersensitive response caused by basidiospore-derived infection of the cowpea rust fungus

The hypersensitive response (HR) of disease-resistant plant cells to fungal invasion is a rapid cell death that has some features in common with programmed cell death (apoptosis) in animals. We investigated the role of cytosolic free calcium ([Ca2+]i) in the HR of cowpea to the cowpea rust fungus. By using confocal laser scanning microscopy in conjunction with a calcium reporter dye, we found a slow, prolonged elevation of [Ca2+]i in epidermal cells of resistant but not susceptible plants as the fungus grew through the cell wall. [Ca2+]i levels declined to normal levels as the fungus entered and grew within the cell lumen. This elevation was related to the stage of fungal growth and not to the speed of initiation of subsequent cell death. Elevated [Ca2+]i levels also represent the first sign of the HR detectable in this cowpea-cowpea rust fungus system. The increase in [Ca2+]i was prevented by calcium channnel inhibitors. This effect was consistent with pharmacological tests in which these inhibitors delayed the HR. The data suggest that elevation of [Ca2+]i is involved in signal transduction leading to the HR during rust fungal infection.     

Rust secreted protein Ps87 is conserved in diverse fungal pathogens and contains a RXLR-like motif sufficient for translocation into plant cells

Background

Effector proteins of biotrophic plant pathogenic fungi and oomycetes are delivered into host cells and play important roles in both disease development and disease resistance response. How obligate fungal pathogen effectors enter host cells is poorly understood. The Ps87 gene of Puccinia striiformis encodes a protein that is conserved in diverse fungal pathogens. Ps87 homologs from a clade containing rust fungi are predicted to be secreted. The aim of this study is to test whether Ps87 may act as an effector during Puccinia striiformis infection.

Methodology/Principal Findings

Yeast signal sequence trap assay showed that the rust protein Ps87 could be secreted from yeast cells, but a homolog from Magnaporthe oryzae that was not predicted to be secreted, could not. Cell re-entry and protein uptake assays showed that a region of Ps87 containing a conserved RXLR-like motif [K/R]RLTG was confirmed to be capable of delivering oomycete effector Avr1b into soybean leaf cells and carrying GFP into soybean root cells. Mutations in the Ps87 motif (KRLTG) abolished the protein translocation ability.

Conclusions/Significance

The results suggest that Ps87 and its secreted homologs could utilize similar protein translocation machinery as those of oomycete and other fungal pathogens. Ps87 did not show direct suppression activity on plant defense responses. These results suggest Ps87 may represent an “emerging effector” that has recently acquired the ability to enter plant cells but has not yet acquired the ability to alter host physiology.     

Enzymatic specificity of three ribosome-inactivating proteins against fungal ribosomes,and correlation with antifungal activity

Ribosome-inactivating proteins (RIPs) are enzymes that cleave a specific adenine base from the highly conserved sarcin/ricin (S/R) loop of the large ribosomal RNA, thus arresting protein synthesis at the translocation step. In the present study, we employed three RIPs to dissect the antifungal activity of RIPs as plant defense proteins. We measured the catalytic activity of RAT (the catalytic A-chain of ricin from Ricinus communis L.), saporin-S6 (from Saponaria officinalis L.), and ME (RIP from Mirabilis expansa R&P) against intact ribosomal substrates isolated from various pathogenic fungi. We further determined the enzymatic specificity of these three RIPs against fungal ribosomes, from Rhizoctonia solani Kuhn, Alternaria solani Sorauer, Trichoderma reesei Simmons and Candida albicans Berkhout, and correlated the data with antifungal activity. RAT showed the strongest toxicity against all tested fungal ribosomes, except for the ribosomes isolated from C. albicans, which were most susceptible to saporin. RAT and saporin showed higher enzymatic activity than ME against ribosomes from all of the fungal species assayed, but did not show detectable antifungal activity. In contrast, ME showed substantial inhibitory activity against fungal growth. Using N-hydroxysuccinimide-fluorescein labeling of RIPs and fluorescence microscopy, we determined that ME was targeted to the surface of fungal cells and transferred into the cells. Thus, ME caused ribosome depurination and subsequent fungal mortality. In contrast, saporin did not interact with fungal cells, correlating with its lack of antifungal activity.     

Fungal elicitor-induced retardation and its restoration of root growth in tobacco seedlings

In this study, we investigated responses of growing and intact tobacco (N. tabacum cv Xanthi) seedlings to a fungal elicitor, a xylanase from Trichoderma viride (TvX). In addition to the induction of defense gene expression, TvX treatment caused the retardation of growth of seedlings. In the TvX-treated seedlings, growth of primary roots was markedly reduced through repression of cell division and longitudinal cell elongation in a meristematic zone and an elongation zone, respectively. However, cell differentiation to form vascular bundles and root hairs continued. In the TvX-treated root cap, disappearance of starch granules in columella cells and aggregation of border cells were observed. Furthermore, the TvX-induced growth retardation was restored after removal of the elicitor, resulting in a plastic alteration of root architecture. Therefore, the fungal elicitor might act as an environmental cue that regulates root growth and development as well as the ordinary defense responses in plant seedlings. These findings suggest a novel aspect of plant growth regulation via a plant–microbe interaction in the rhizosphere.     

Expression of fungal pectin methylesterase in transgenic tobacco leads to alteration in cell wall metabolism and a dwarf phenotype

A transgenic tobacco plant (Nicotiana tabacum L.) expressing a fungal pectin methylesterase (PME; EC 3.1.1.11) gene derived from a black filamentous fungus, Aspergillus niger was created. Fungal PME should have a wider range of adaptability to substrate pectin compared with plant PME. As expected, the proportion of methyl esters in pectin was reduced in the transgenic tobacco. Consequently, the transgenic plant showed short internodes, small leaves and a dwarf phenotype. At a cellular level, the longitudinal lengths of stem epidermal cells were shorter than those of control plants. This is the first report that fungal PME promotes dwarfism in plants. It is worth noting that in the PME-expressing dwarf plant, the expression levels of cell wall metabolism related genes that included endo-1,4-beta-glucanase, cellulose synthase, endo-xyloglucan transferase and expansin gene were decreased. These results suggest that the expression of fungal PME in plants affects the cell wall metabolism.     

Oomycete and fungal effector entry, a microbial Trojan horse

Oomycete and fungal symbionts have significant impacts on most commercially important crop and forest species, and on natural ecosystems, both negatively as pathogens and positively as mutualists. Symbiosis may be facilitated through the secretion of effector proteins, some of which modulate a variety of host defense mechanisms. A subset of these secreted proteins are able to translocate into host cells. In the oomycete pathogens, two conserved N-terminal motifs, RXLR and dEER, mediate translocation of effector proteins into host cells independent of any pathogen-encoded machinery. An expanded 'RXLR-like' motif [R/K/H]X[L/M/I/F/Y/W]X has been used to identify functional translocation motifs in host-cell-entering fungal effector proteins from pathogens and a mutualist. The RXLR-like translocation motifs were required for the fungal effectors to enter host cells in the absence of any pathogen-encoded machinery. Oomycete and fungal effectors with RXLR and RXLR-like motifs can bind phospholipids, specifically phosphatidylinositol-3-phosphate (PtdIns-3-P). Effector-PtdIns-3-P binding appears to mediate cell entry via lipid raft-mediated endocytosis, and could be blocked by sequestering cell surface PtdIns-3-P or by utilizing inositides that competitively inhibit effector binding to PtdIns-3-P. These findings suggest that effector blocking technologies could be developed and utilized in a variety of important crop species against a broad spectrum of plant pathogens.     

Cell polarization,a crucial process in fungal defence

Plant cells responding to fungal attack undergo large morphological alterations, along with rapid and extensive metabolic reprogramming. Cytological analysis of single infected plant cells revealed a large complexity of interdependent, rapid and dynamic changes of a multitude of cellular components. Among these changes are major rearrangements of the cytoskeleton, translocation of cytoplasm and of the cell nucleus to the fungal penetration site, and local apposition of barrier material around this site, which results in massive cell-wall reinforcement. If this first line of defence is overcome by the pathogen, in many cases, it is followed by hypersensitive plant cell death, which stops growth of the penetrating fungus and finally leads to its death. The speed and magnitude of the initial defence response appear to be crucial to plant disease resistance.     

Cleavage of the Pseudomonas syringae type III effector AvrRpt2 requires a host factor(s) common among eukaryotes and is important for AvrRpt2 localization in the host cell

Many phytopathogenic bacteria use a type III secretion system to deliver type III effector proteins into the host plant cell. The Pseudomonas syringae type III effector AvrRpt2 is cleaved at a specific site when translocated into the host cell. In this study, we first demonstrate that the factor(s) required for AvrRpt2 cleavage is present in extracts from animal and yeast cells, as well as plant cells. The cleavage factor in animal and plant cell extracts was heat labile but relatively insensitive to protease inhibitors. Second, mutational analysis of AvrRpt2 was applied to identify features important for its cleavage. In addition to two of the amino acid residues in the immediate vicinity of the cleavage site, a large part of the region C-terminal to the cleavage site was required when AvrRpt2 was cleaved in animal cell extract. Most of these features were also important when AvrRpt2 was cleaved in plant cells. Third, we investigated the effect of cleavage in interactions of AvrRpt2 with plant cells. Cleavage of AvrRpt2 appeared to be important for proper interactions with Arabidopsis cells that lack the resistance gene product corresponding to AvrRpt2, RPS2. In addition, removal of the region N-terminal to the cleavage site was important for the correct localization of the C-terminal effector region of the protein in the host cell. We speculate that the virulence function of AvrRpt2 requires removal of the N-terminal region to redirect the effector protein to a specific subcellular location in the host cell after translocation of the protein.     

Cloning and expression analysis of Phytoplasma protein translocation genes

Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.     

Translocation of cell‐penetrating peptides into Candida fungal pathogens

Cell‐penetrating peptides (CPPs) are small peptides capable of crossing cellular membranes while carrying molecular cargo. Although they have been widely studied for their ability to translocate nucleic acids, small molecules, and proteins into mammalian cells, studies of their interaction with fungal cells are limited. In this work, we evaluated the translocation of eleven fluorescently labeled peptides into the important human fungal pathogens Candida albicans and C. glabrata and explored the mechanisms of translocation. Seven of these peptides (cecropin B, penetratin, pVEC, MAP, SynB, (KFF)₃K, and MPG) exhibited substantial translocation (>80% of cells) into both species in a concentration‐dependent manner, and an additional peptide (TP‐10) exhibiting strong translocation into only C. glabrata. Vacuoles were involved in translocation and intracellular trafficking of the peptides in the fungal cells and, for some peptides, escape from the vacuoles and localization in the cytosol were correlated to toxicity toward the fungal cells. Endocytosis was involved in the translocation of cecropin B, MAP, SynB, MPG, (KFF)₃K, and TP‐10, and cecropin B, penetratin, pVEC, and MAP caused membrane permeabilization during translocation. These results indicate the involvement of multiple translocation mechanisms for some CPPs. Although high levels of translocation were typically associated with toxicity of the peptides toward the fungal cells, SynB was translocated efficiently into Candida cells at concentrations that led to minimal toxicity. Our work highlights the potential of CPPs in delivering antifungal molecules and other bioactive cargo to Candida pathogens.     

Signalling between Pathogenic Rust Fungi and Resistant or Susceptible Host Plants

Rust fungi are obligately biotrophic plant parasites that obtaintheir nutrients from living host cells. The initiation of thetwo parasitic phases of these fungi generally requires topographicsignals from the plant surface followed, for the dikaryoticphase, by a successive sequence of signals to control furtherfungal development within the plant. During the fungal lifecycle, three types of intracellular structures (invasion hyphae,M-, and D-haustoria) are formed and each may differently affectthe host membrane that surrounds it, as well as affecting othercellular components. Each intracellular structure also preventsnon-specific plant defences triggered by fungal activities,possibly by interfering with the signalling system rather thandefence expression. In resistant host cultivars, cellular invasiontriggers a rapid cell death (the hypersensitive response) thatshares some features with developmentally programmed cell deathin animal and plant tissues, and is controlled by parasite-specificresistance genes that resemble those that defend plants againstother types of pathogens. Evidence from one system suggeststhat this response is specifically elicited by a fungal peptideand does not involve the oxidative burst typical of resistanceexpression in other plant-pathogen interactions. However, overall,few of the molecules involved in any of these plant-rust fungiinteractions have been completely characterized and much isleft to be discovered, particularly with respect to how cellularsusceptibility to rust fungi is conditioned.Copyright 1997 Annalsof Botany Company Apoptosis; biotrophy; elicitor; hypersensitive response; oxidative burst; suppressor; Uromyces vignae     

At the poles across kingdoms: phosphoinositides and polar tip growth

Phosphoinositides (PIs) are minor, but essential phospholipid constituents of eukaryotic membranes, and are involved in the regulation of various physiological processes. Recent genetic and cell biological advances indicate that PIs play important roles in the control of polar tip growth in plant cells. In root hairs and pollen tubes, PIs control directional membrane trafficking required for the delivery of cell wall material and membrane area to the growing tip. So far, the exact mechanisms by which PIs control polarity and tip growth are unresolved. However, data gained from the analysis of plant, fungal and animal systems implicate PIs in the control of cytoskeletal dynamics, ion channel activity as well as vesicle trafficking. The present review aims at giving an overview of PI roles in eukaryotic cells with a special focus on functions pertaining to the control of cell polarity. Comparative screening of plant and fungal genomes suggests diversification of the PI system with increasing organismic complexity. The evolutionary conservation of the PI system among eukaryotic cells suggests a role for PIs in tip growing cells in models where PIs so far have not been a focus of attention, such as fungal hyphae.     

Direct plant gene delivery with a poly(amidoamine) dendrimer

Plant gene delivery is challenging due to the presence of plant cell walls. Conventional means such as Agrobacterium infection, biolistic particle bombardment, electroporation, or polyethylene glycol attachment are often characterized by high cost, labor extensiveness, and a significant perturbation to the growth of cells. We have succeeded in delivering GFP-encoding plasmid DNA to turfgrass cells using poly(amidoamine) dendrimers. Our new scheme utilizes the physiochemical properties as well as the nanosize of the poly(amidoamine) dendrimer for direct and noninvasive gene delivery. The GFP gene was expressed in the plant cells as observed by confocal fluorescence microscopy. The transfection efficiency may be further improved by optimizing the pH of the cell culture medium and the molar ratio of the dendrimer to DNA. The use of the current delivery system can be extended to virtually all plant species having successful regeneration systems in place.