Chenlei Hua

A Phytophthora sojae G-protein alpha subunit is involved in chemotaxis to soybean isoflavones.

ABSTRACT For the soybean pathogen Phytophthora sojae, chemotaxis of zoospores to isoflavones is believed to be critical for recognition of the host and for initiating infection. However, the molecular mechanisms underlying this chemotaxis are largely unknown. To investigate the role of G-protein and calcium signaling in chemotaxis, we analyzed the expression of several genes known to be involved in these pathways and selected one that was specifically expressed in sporangia and zoospores but not in mycelium. This gene, named PsGPA1, is a single-copy gene in P. sojae and encodes a G-protein α subunit that shares 96% identity in amino acid sequence with that of Phytophthora infestans. To elucidate the function, expression of PsGPA1 was silenced by introducing antisense constructs into P. sojae. PsGPA1 silencing did not disturb hyphal growth or sporulation but severely affected zoospore behavior, including chemotaxis to the soybean isoflavone daidzein. Zoospore encystment and cyst germination were also altered, resulting in the inability of the PsGPA1-silenced mutants to infect soybean. In addition, the expressions of a calmodulin gene, PsCAM1, and two calcium- and calmodulin-dependent protein kinase genes, PsCMK3 and PsCMK4, were increased in the mutant zoospores, suggesting that PsGPA1 negatively regulates the calcium signaling pathways that are likely involved in zoospore chemotaxis.

GK4, a G‐protein‐coupled receptor with a phosphatidylinositol phosphate kinase domain in Phytophthora infestans, is involved in sporangia development and virulence

For dispersal and host infection plant pathogens largely depend on asexual spores. Pathogenesis and sporulation are complex processes that are governed by cellular signalling networks including G‐protein and phospholipid signalling. Oomycetes possess a family of novel proteins called GPCR‐PIPKs (GKs) that are composed of a seven‐transmembrane spanning (7‐TM) domain fused to a phosphatidylinositol phosphate kinase (PIPK) domain. Based on this domain structure GKs are anticipated to link G‐protein and phospholipid signal pathways; however, their functions are currently unknown. Expression analyses of the 12 GK genes in Phytophthora infestans and their orthologues in Phytophthora sojae, revealed differential expression during asexual development. PiGK1 and PiGK4 were fused to monomeric red fluorescent protein (mRFP) and ectopically expressed in P. infestans. In growing hyphae different subcellular distribution patterns were observed indicating that these two GKs act independently during development. We focused on the functional analyses of PiGK4. Its localization suggested involvement in cell differentiation and elongation and its 7‐TM domain showed a canonical GPCR membrane topology. Silencing of GK4 and overexpression of full‐length and truncated constructs in P. infestans revealed that PiGK4 is not only involved in spore germination and hyphal elongation but also in sporangia cleavage and infection.

Genome-wide identification of Phytophthora sojae SNARE genes and functional characterization of the conserved SNARE PsYKT6.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are central components of the machinery mediating membrane fusion and key factors for vesicular trafficking in all eukaryotic cells. Taking advantage of the available whole genome sequence of the oomycete plant pathogen Phytophthora sojae, 35 genes encoding putative SNARE proteins were identified in the genome of this organism. PsYKT6, one of the most conserved SNARE proteins, was functionally characterized by homology-dependent gene silencing. The phenotype analysis showed that PsYKT6 is important for proper asexual development, sexual reproduction, and pathogenesis on host soybean cultivars.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: Identification and functional analysis of putative D-galacturonate transporters

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

Chemotaxis and oospore formation in Phytophthora sojae are controlled by G-protein-coupled receptors with a phosphatidylinositol phosphate kinase domain

G‐protein‐coupled receptors (GPCRs) are key cellular components that mediate extracellular signals into intracellular responses. Genome mining revealed that Phytophthora spp. have over 60 GPCR genes among which a prominent class of 12 encoding novel proteins with an N‐terminal GPCR domain fused to a C‐terminal phosphatidylinositol phosphate kinase (PIPK) domain. This study focuses on two GPCR‐PIPKs (GKs) in Phytophthora sojae. PsGK4 and PsGK5 are differentially expressed during the life cycle with the highest expression in cysts and during cyst germination, and at late infection stages. In P. sojae transformants that constitutively express RFP‐tagged PsGK4 and PsGK5, the fusion proteins in hyphae reside in small, rapidly moving vesicular‐like structures. Functional analysis using gene silencing showed that PsGK4‐silenced transformants displayed higher levels of encystment and a reduced cyst germination rate when compared with the recipient strain. Moreover, GK4 deficiency (or reduction) resulted in severe defects in zoospore chemotaxis towards isoflavones and soybean roots. In contrast, PsGK5‐silenced transformants exhibited no obvious defects in asexual development but oospore production was severely impaired. Both, PsGK4‐ and PsGK5‐silenced transformants showed reduced pathogenicity. These results point to involvement of GKs in zoospore behaviour, chemotaxis and oospore development, and suggest that PsGK4 and PsGK5 each head independent signalling pathways.

Actin dynamics in Phytophthora infestans; rapidly reorganizing cables and immobile, long-lived plaques

The actin cytoskeleton is a dynamic but well‐organized intracellular framework that is essential for proper functioning of eukaryotic cells. Here, we use the actin binding peptide Lifeact to investigate the in vivo actin cytoskeleton dynamics in the oomycete plant pathogen Phytophthora infestans. Lifeact–eGFP labelled thick and thin actin bundles and actin filament plaques allowing visualization of actin dynamics. All actin structures in the hyphae were cortically localized. In growing hyphae actin filament cables were axially oriented in the sub‐apical region whereas in the extreme apex in growing hyphae, waves of fine F‐actin polymerization were observed. Upon growth termination, actin filament plaques appeared in the hyphal tip. The distance between a hyphal tip and the first actin filament plaque correlated strongly with hyphal growth velocity. The actin filament plaques were nearly immobile with average lifetimes exceeding 1 h, relatively long when compared to the lifetime of actin patches known in other eukaryotes. Plaque assembly required ∼30 s while disassembly was accomplished in ∼10 s. Remarkably, plaque disassembly was not accompanied with internalization and the formation of endocytic vesicles. These findings suggest that the functions of actin plaques in oomycetes differ from those of actin patches present in other organisms.

The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst

In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P. sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.

Effect of Flumorph on F-Actin Dynamics in the Potato Late Blight Pathogen Phytophthora infestans

Oomycetes are fungal-like pathogens that cause notorious diseases. Protecting crops against oomycetes requires regular spraying with chemicals, many with an unknown mode of action. In the 1990s, flumorph was identified as a novel crop protection agent. It was shown to inhibit the growth of oomycete pathogens including Phytophthora spp., presumably by targeting actin. We recently generated transgenic Phytophthora infestans strains that express Lifeact-enhanced green fluorescent protein (eGFP), which enabled us to monitor the actin cytoskeleton during hyphal growth. For analyzing effects of oomicides on the actin cytoskeleton in vivo, the P. infestans Lifeact-eGFP strain is an excellent tool. Here, we confirm that flumorph is an oomicide with growth inhibitory activity. Microscopic analyses showed that low flumorph concentrations provoked hyphal tip swellings accompanied by accumulation of actin plaques in the apex, a feature reminiscent of tips of nongrowing hyphae. At higher concentrations, swelling was more pronounced and accompanied by an increase in hyphal bursting events. However, in hyphae that remained intact, actin filaments were indistinguishable from those in nontreated, nongrowing hyphae. In contrast, in hyphae treated with the actin depolymerizing drug latrunculin B, no hyphal bursting was observed but the actin filaments were completely disrupted. This difference demonstrates that actin is not the primary target of flumorph.

Phytophthora infestans small phospholipase D-like proteins elicit plant cell death and promote virulence: P. infestans small PLD-likes promote virulence

Summary The successful invasion of host tissue by (hemi‐)biotrophic plant pathogens is dependent on modifications of the host plasma membrane to facilitate the two‐way transfer of proteins and other compounds. Haustorium formation and the establishment of extrahaustorial membranes are probably dependent on a variety of enzymes that modify membranes in a coordinated fashion. Phospholipases, enzymes that hydrolyse phospholipids, have been implicated as virulence factors in several pathogens. The oomycete Phytophthora infestans is a hemibiotrophic pathogen that causes potato late blight. It possesses different classes of phospholipase D (PLD) proteins, including small PLD‐like proteins with and without signal peptide (sPLD‐likes and PLD‐likes, respectively). Here, we studied the role of sPLD‐like‐1, sPLD‐like‐12 and PLD‐like‐1 in the infection process. They are expressed in expanding lesions on potato leaves and during in vitro growth, with the highest transcript levels in germinating cysts. When expressed in planta in the presence of the silencing suppressor P19, all three elicited a local cell death response that was visible at the microscopic level as autofluorescence and strongly boosted in the presence of calcium. Moreover, inoculation of leaves expressing the small PLD‐like genes resulted in increased lesion growth and greater numbers of sporangia, but this was abolished when mutated PLD‐like genes were expressed with non‐functional PLD catalytic motifs. These results show that the three small PLD‐likes are catalytically active and suggest that their enzymatic activity is required for the promotion of virulence, possibly by executing membrane modifications to support the growth of P. infestans in the host.