A Phytophthora capsici RXLR effector manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein trafficking pathway

The oomycete pathogen Phytophthora capsici encodes hundreds of RXLR effectors to enter plant cells and suppress host defense responses. Only few of them are conserved across different strains and species. Such ‘core effectors’ may target hub immunity pathways that are essential during Phytophthora pathogens interacting with their hosts. However, the underlying mechanisms of core RXLRs-mediated host immunity manipulation are largely unknown. Here, we report the functional characterization of a P. capsici RXLR effector, RXLR242. RXLR242 expression is highly induced during the infection process. Its ectopic expression in Nicotiana benthamiana promotes Phytophthora infection. RXLR242 physically interacts with a group of RAB proteins, which belong to the small GTPase family and function in specifying transport pathways in the intracellular membrane trafficking system. RXLR242 impedes the secretion of PATHOGENESIS-RELATED 1 (PR1) protein to the apoplast by interfering the formation of RABE1-7-labeled vesicles. Further analysis indicated that such phenomenon is resulted from competitive binding of RXLR242 to RABE1-7. RXLR242 also interferes trafficking of the membrane-located receptor FLAGELLIN-SENSING 2 (FLS2) through competitively interacting with RABA4-3. Taken together, our work demonstrates that RXLR242 manipulates plant immunity by targeting RAB proteins and disturbing vesicle-mediated protein transporting pathway in plant hosts.

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The RXLR Effector PcAvh1 Is Required for Full Virulence of Phytophthora capsici

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-09-18-0251-r ◽

2019 ◽

Vol 32 (8) ◽

pp. 986-1000 ◽

Cited By ~ 9

Author(s):

Xiao-Ren Chen ◽

Ye Zhang ◽

Hai-Yang Li ◽

Zi-Hui Zhang ◽

Gui-Lin Sheng ◽

...

Keyword(s):

Plant Pathogens ◽

Plant Immunity ◽

Phytophthora Capsici ◽

Ectopic Expression ◽

Plant Cells ◽

Effector Proteins ◽

Bell Pepper ◽

Broad Host Range ◽

Virus Induced Gene Silencing ◽

Rxlr Effector

Plant pathogens employ diverse secreted effector proteins to manipulate host physiology and defense in order to foster diseases. The destructive Phytophthora pathogens encode hundreds of cytoplasmic effectors, which are believed to function inside the plant cells. Many of these cytoplasmic effectors contain the conserved N-terminal RXLR motif. Understanding the virulence function of RXLR effectors will provide important knowledge of Phytophthora pathogenesis. Here, we report the characterization of RXLR effector PcAvh1 from the broad–host range pathogen Phytophthora capsici. Only expressed during infection, PcAvh1 is quickly induced at the early infection stages. CRISPR/Cas9-knockout of PcAvh1 in P. capsici severely impairs virulence while overexpression enhances disease development in Nicotiana benthamiana and bell pepper, demonstrating that PcAvh1 is an essential virulence factor. Ectopic expression of PcAvh1 induces cell death in N. benthamiana, tomato, and bell pepper. Using yeast two-hybrid screening, we found that PcAvh1 interacts with the scaffolding subunit of the protein phosphatase 2A (PP2Aa) in plant cells. Virus-induced gene silencing of PP2Aa in N. benthamiana attenuates resistance to P. capsici and results in dwarfism, suggesting that PP2Aa regulates plant immunity and growth. Collectively, these results suggest that PcAvh1 contributes to P. capsici infection, probably through its interaction with host PP2Aa.

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A small cysteine-rich phytotoxic protein of Phytophthora capsici functions as both plant defense elicitor and virulence factor

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-21-0025-r ◽

2021 ◽

Author(s):

Zi-Hui Zhang ◽

Jinghao Jin ◽

Gui-Lin Sheng ◽

Yu-Ping Xing ◽

Wang Liu ◽

...

Keyword(s):

Plant Defense ◽

Virulence Factor ◽

Phytophthora Capsici ◽

Ectopic Expression ◽

Defense Responses ◽

Phytophthora Species ◽

Plant Interactions ◽

Defense Gene Expression ◽

Callose Deposition ◽

Defense Gene

Small cysteine-rich (SCR) proteins including fungal avirulence proteins play important roles in the pathogen-plant interactions. SCR protein-encoding genes have been discovered in the genomes of Phytophthora pathogens, but their functions during the pathogenesis remain obscure. Here, we report the characterization of one Phytophthora capsici SCR protein, namely SCR82 with similarity to Phytophthora cactorum phytotoxic protein PcF. The scr82 gene has 10 allelic sequences in the P. capsici population. Homologues of SCR82 were not identified in fungi or other organisms but in Phytophthora relative species. Initially scr82 was weakly expressed during the mycelium, sporangium and zoospore stages, but quickly upregulated when the infection initiated. Both ectopic expression of SCR82 and recombinant yeast-expressed protein (rSCR82) caused cell death on tomato leaves. Upon treatment, rSCR82 induced plant defense responses including the induction of defense gene expression, reactive oxygen species burst and callose deposition. Knockout of scr82 in P. capsici by CRISPR/Cas9 severely impaired its virulence on host plants and reduced significantly its resistance againstoxidative stress. Inversely, its overexpression increased the pathogen’s virulence and tolerance to oxidative stress. Our results collectively demonstrate that SCR82 functions as both an important virulence factor and plant defense elicitor, which is conserved across Phytophthora species.

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Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients

Blood ◽

10.1182/blood-2002-09-2789 ◽

2003 ◽

Vol 101 (7) ◽

pp. 2736-2742 ◽

Cited By ~ 65

Author(s):

Gaël Ménasché ◽

Jérôme Feldmann ◽

Anne Houdusse ◽

Catherine Desaymard ◽

Alain Fischer ◽

...

Keyword(s):

Functional Characterization ◽

Nucleotide Binding ◽

Binding Activity ◽

Small Gtpase ◽

Rab Proteins ◽

Missense Mutations ◽

Immune Disorder ◽

Griscelli Syndrome ◽

Protein Functions ◽

Cytotoxic Granule

Rab27a is a member of the Rab family of small GTPase proteins, and thus far is the first member to be associated with a human disease (ie, the Griscelli syndrome type 2). Mutations in the Rab27a gene cause pigment as well as cytotoxic granule transport defects, accounting for the partial albinism and severe immune disorder characteristics of this syndrome. So far, 3 Rab27a missense mutations have been identified. They open a unique opportunity to designate critical structural and functional residues of Rab proteins. We show here that the introduction of a proline residue in the α4 (Ala152Pro) or β5 (Leu130Pro) loop, observed in 2 of these spontaneous mutants, dramatically affects both guanosine triphosphate (GTP) and guanosine diphosphate (GDP) nucleotide-binding activity of Rab27a, probably by disrupting protein folding. The third mutant, Trp73Gly, is located within an invariant hydrophobic triad at the switch interface, and was previously shown in active Rab3A to mediate rabphilin3A effector interaction. Trp73Gly is shown to display the same nucleotide-binding and GTPase characteristics as the constitutively active mutant Gln78Leu. However, in contrast to Gln78Leu, Trp73Gly mutant construct neither interacts with the Rab27a effector melanophilin nor modifies melanosome distribution and cytotoxic granule exocytosis. Substitutions introduced at the 73 position, including the leucine residue present in Ras, did not restore Rab27a protein functions. Taken together, our results characterize new critical residues of Rab proteins, and identify the Trp73 residue of Rab27a as a key position for interaction with the specific effectors of Rab27a, both in melanocytes and cytotoxic cells.

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Human Rab small GTPase- and class V myosin-mediated membrane tethering in a chemically defined reconstitution system

10.1101/129072 ◽

2017 ◽

Author(s):

Motoki Inoshita ◽

Joji Mima

Keyword(s):

Lipid Bilayers ◽

Membrane Trafficking ◽

Small Gtpases ◽

Small Gtpase ◽

Effector Proteins ◽

Rab Proteins ◽

Dependent Manner ◽

Class V ◽

Rab Family ◽

Membrane Tethering

AbstractMembrane tethering is a fundamental process essential for compartmental specificity of intracellular membrane trafficking in eukaryotic cells. Rab-family small GTPases and specific sets of Rab-interacting effector proteins, including coiled-coil tethering proteins and multisubunit tethering complexes, have been reported to be responsible for membrane tethering. However, whether and how these key components directly and specifically tether subcellular membranes still remains enigmatic. Using chemically defined proteoliposomal systems reconstituted with purified human Rab proteins and synthetic liposomal membranes to study the molecular basis of membrane tethering, we established here that Rab-family GTPases have a highly conserved function to directly mediate membrane tethering, even in the absence of any types of Rab effectors such as the so-called tethering proteins. Moreover, we demonstrate that membrane tethering mediated by endosomal Rab11a is drastically and selectively stimulated by its cognate Rab effectors, class V myosins (Myo5A and Myo5B), in a GTP-dependent manner. Of note, Myo5A and Myo5B exclusively recognized and cooperated with the membrane-anchored form of their cognate Rab11a to support membrane tethering mediated by trans-Rab assemblies on apposing membranes. Our findings support the novel concept that Rab-family proteins provide a bona fide membrane tether to physically and specifically link two distinct lipid bilayers of subcellular membranes. They further indicate that Rab-interacting effector proteins, including class V myosins, can regulate these Rab-mediated membrane tethering reactions.

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Ectopic expression of LhMYC2 increases susceptibility to Botrytis cinerea in Arabidopsis thaliana

Canadian Journal of Plant Science ◽

10.1139/cjps-2020-0047 ◽

2020 ◽

Author(s):

Qi Cui ◽

Xue Gao ◽

Lianjuan Wang ◽

Guixia Jia

Keyword(s):

Functional Characterization ◽

Ectopic Expression ◽

Defense Responses ◽

Gray Mold ◽

Wild Type ◽

Transcriptional Changes ◽

Negative Role ◽

Ja Signaling ◽

Fungal Inoculation

Gray mold disease, mainly caused by Botrytis elliptica and B. cinerea, leads to severe losses in lily cut flower and bulb production. MYC2 is a critical regulator of the activation of jasmonate (JA)-mediated defense responses in plants. However, information about the lily MYC2 gene is limited. Therefore, functional characterization of MYC2 in lily, especially its role in plant immune responses, should be performed. Here, significant differences between the Botrytis-resistant Lilium hybrid ‘Sorbonne’ and the Botrytis-susceptible ‘Tresor’ were found following B. cinerea inoculation, as indicated by jasmonic acid (JA) and JA-isoleucine (JA-Ile) accumulation and related gene expression. More JA and JA-Ile were detected in ‘Sorbonne’ than in ‘Tresor’ following fungal inoculation, and higher transcript levels of JA biosynthesis genes (LhAOS, LhAOC, and LhOPR3) and a signaling gene (LhCOI1) were detected in ‘Sorbonne’ than in ‘Tresor’. In contrast, expression of the critical signaling regulator LhMYC2 was higher in ‘Tresor’ than in ‘Sorbonne’. LhMYC2 was then isolated from ‘Sorbonne’ and found to be similar to several plant MYC2 homologs that have pivotal roles in JA signaling. The expression of LhMYC2 increased significantly in response to JA and salicylic acid (SA) in ‘Sorbonne’. Ectopic expression of LhMYC2 in Arabidopsis resulted in greater susceptibility to B. cinerea than that observed in wild-type plants. This susceptibility was coupled with the transcriptional changes in SA- and JA-responsive genes. Overall, our findings indicate that LhMYC2 plays a negative role in Arabidopsis resistance to B. cinerea.

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A Phytophthora capsici RXLR effector targets and inhibits the central immune kinases to suppress plant immunity

New Phytologist ◽

10.1111/nph.17573 ◽

2021 ◽

Author(s):

Xiangxiu Liang ◽

Yazhou Bao ◽

Meixiang Zhang ◽

Dandan Du ◽

Shaofei Rao ◽

...

Keyword(s):

Plant Immunity ◽

Phytophthora Capsici ◽

Rxlr Effector

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An Arabidopsis downy mildew non-RxLR effector suppresses induced plant cell death to promote biotroph infection

Journal of Experimental Botany ◽

10.1093/jxb/eraa472 ◽

2020 ◽

Author(s):

Florian Dunker ◽

Lorenz Oberkofler ◽

Bernhard Lederer ◽

Adriana Trutzenberg ◽

Arne Weiberg

Keyword(s):

Cell Death ◽

Pseudomonas Syringae ◽

Plant Cell ◽

Phytophthora Capsici ◽

Ectopic Expression ◽

Molecular Evidence ◽

Disease Symptoms ◽

Rxlr Effectors ◽

Rxlr Effector ◽

Local Plant

Abstract Our understanding of obligate biotrophic pathogens is limited by lack of knowledge concerning the molecular function of virulence factors. We established Arabidopsis host-induced gene silencing (HIGS) to explore gene functions of Hyaloperonospora arabidopsidis, including CYSTEINE-RICH PROTEIN (HaCR)1, a potential secreted effector gene of this obligate biotrophic pathogen. HaCR1 HIGS resulted in H. arabidopsidis-induced local plant cell death and reduced pathogen reproduction. We functionally characterized HaCR1 by ectopic expression in Nicotiana benthamiana. HaCR1 was capable of inhibiting effector-triggered plant cell death. Consistent with this, HaCR1 expression in N. benthamiana led to stronger disease symptoms caused by the hemibiotrophic oomycete pathogen Phytophthora capsici, but reduced disease symptoms caused by the necrotrophic fungal pathogen Botrytis cinerea. Expressing HaCR1 in transgenic Arabidopsis confirmed higher susceptibility to H. arabidopsidis and to the bacterial hemibiotrophic pathogen Pseudomonas syringae. Increased H. arabidopsidis infection was in accordance with reduced PATHOGENESIS RELATED (PR)1 induction. Expression of full-length HaCR1 was required for its function, which was lost if the signal peptide was deleted, suggesting its site of action in the plant apoplast. This study provides phytopathological and molecular evidence for the importance of this widespread, but largely unexplored class of non-RxLR effectors in biotrophic oomycetes.

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Novel and Converging Ways of NOX2 and SOD3 in Trafficking and Redox Signaling in Macrophages

Antioxidants ◽

10.3390/antiox10020172 ◽

2021 ◽

Vol 10 (2) ◽

pp. 172

Author(s):

Steen Vang Petersen ◽

Nanna Bach Poulsen ◽

Cecilie Linneberg Matthiesen ◽

Frederik Vilhardt

Keyword(s):

Hydrogen Peroxide ◽

Membrane Trafficking ◽

Spatial Organization ◽

Redox Signaling ◽

Small Gtpase ◽

Paracrine Signaling ◽

Tissue Macrophage ◽

Storage Vesicles ◽

Superoxide Dismutase 3 ◽

Macrophage Populations

Macrophages and related tissue macrophage populations use the classical NADPH oxidase (NOX2) for the regulated production of superoxide and derived oxidants for pathogen combat and redox signaling. With an emphasis on macrophages, we discuss how sorting into secretory storage vesicles, agonist-responsive membrane trafficking, and segregation into sphingolipid and cholesterol-enriched microdomains (lipid rafts) determine the subcellular distribution and spatial organization of NOX2 and superoxide dismutase-3 (SOD3). We discuss how inflammatory activation of macrophages, in part through small GTPase Rab27A/B regulation of the secretory compartments, mediates the coalescence of these two proteins on the cell surface to deliver a focalized hydrogen peroxide output. In interplay with membrane-embedded oxidant transporters and redox sensitive target proteins, this arrangement allows for the autocrine and paracrine signaling, which govern macrophage activation states and transcriptional programs. By discussing examples of autocrine and paracrine redox signaling, we highlight why formation of spatiotemporal microenvironments where produced superoxide is rapidly converted to hydrogen peroxide and conveyed immediately to reach redox targets in proximal vicinity is required for efficient redox signaling. Finally, we discuss the recent discovery of macrophage-derived exosomes as vehicles of NOX2 holoenzyme export to other cells.

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Method for the Production and Purification of Plant Immuno-Active Xylanase from Trichoderma

International Journal of Molecular Sciences ◽

10.3390/ijms22084214 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4214

Author(s):

Gautam Anand ◽

Meirav Leibman-Markus ◽

Dorin Elkabetz ◽

Maya Bar

Keyword(s):

Immune System ◽

Plant Defense ◽

Plant Immunity ◽

Xylanase Activity ◽

Hydrolytic Enzymes ◽

Adaptive Immune System ◽

Defense Responses ◽

Plant Defense Responses ◽

Xylanase Production ◽

Ideal System

Plants lack a circulating adaptive immune system to protect themselves against pathogens. Therefore, they have evolved an innate immune system based upon complicated and efficient defense mechanisms, either constitutive or inducible. Plant defense responses are triggered by elicitors such as microbe-associated molecular patterns (MAMPs). These components are recognized by pattern recognition receptors (PRRs) which include plant cell surface receptors. Upon recognition, PRRs trigger pattern-triggered immunity (PTI). Ethylene Inducing Xylanase (EIX) is a fungal MAMP protein from the plant-growth-promoting fungi (PGPF)–Trichoderma. It elicits plant defense responses in tobacco (Nicotiana tabacum) and tomato (Solanum lycopersicum), making it an excellent tool in the studies of plant immunity. Xylanases such as EIX are hydrolytic enzymes that act on xylan in hemicellulose. There are two types of xylanases: the endo-1, 4-β-xylanases that hydrolyze within the xylan structure, and the β-d-xylosidases that hydrolyze the ends of the xylan chain. Xylanases are mainly synthesized by fungi and bacteria. Filamentous fungi produce xylanases in high amounts and secrete them in liquid cultures, making them an ideal system for xylanase purification. Here, we describe a method for cost- and yield-effective xylanase production from Trichoderma using wheat bran as a growth substrate. Xylanase produced by this method possessed xylanase activity and immunogenic activity, effectively inducing a hypersensitive response, ethylene biosynthesis, and ROS burst.

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