scholarly journals Intra-strain elicitation and suppression of plant immunity by Ralstonia solanacearum type-III effectors in Nicotiana benthamiana

2019 ◽  
Author(s):  
Yuying Sang ◽  
Wenjia Yu ◽  
Haiyan Zhuang ◽  
Yali Wei ◽  
Lida Derevnina ◽  
...  
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
Plant Cells ◽  
Effector Proteins ◽  
Type Iii Effectors ◽  
Plant Immune System ◽  
Successful Infection ◽  
Genes Encoding

AbstractEffector proteins delivered inside plant cells are powerful weapons for bacterial pathogens, but this exposes the pathogen to potential recognition by the plant immune system. Therefore, the effector repertoire of a given pathogen must be balanced for a successful infection. Ralstonia solanacearum is an aggressive pathogen with a large repertoire of secreted effectors. One of these effectors, RipE1, is conserved in most R. solanacearum strains sequenced to date. In this work, we found that RipE1 triggers immunity in N. benthamiana, which requires the immune regulator SGT1, but not EDS1 or NRCs. Interestingly, RipE1-triggered immunity induces the accumulation of salicylic acid (SA) and the overexpression of several genes encoding phenylalanine-ammonia lyases (PALs), suggesting that the unconventional PAL-mediated pathway is responsible for the observed SA biosynthesis. Surprisingly, RipE1 recognition also induces the expression of jasmonic acid (JA)-responsive genes and JA biosynthesis, suggesting that both SA and JA may act cooperatively in response to RipE1. Finally, we found that RipE1 expression leads to the accumulation of glutathione in plant cells, which precedes the activation of immune responses. R. solanacearum encodes another effector, RipAY, which is known to inhibit immune responses by degrading cellular glutathione. Accordingly, we show that RipAY inhibits RipE1-triggered immune responses. This work shows a strategy employed by R. solanacearum to counteract the perception of its effector proteins by the plant immune system.

scholarly journals Ralstonia solanacearum Type III Effector RipAY Is a Glutathione-Degrading Enzyme That Is Activated by Plant Cytosolic Thioredoxins and Suppresses Plant Immunity

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Takafumi Mukaihara ◽  
Tadashi Hatanaka ◽  
Masahito Nakano ◽  
Kenji Oda
Keyword(s):  
Immune System ◽  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
Plant Cells ◽  
Effector Proteins ◽  
Yeast Cells ◽  
Yeast Growth ◽  
Type Iii Effector ◽  
Plant Immune System ◽  
Degradation Activity

ABSTRACT The plant pathogen Ralstonia solanacearum uses a large repertoire of type III effector proteins to succeed in infection. To clarify the function of effector proteins in host eukaryote cells, we expressed effectors in yeast cells and identified seven effector proteins that interfere with yeast growth. One of the effector proteins, RipAY, was found to share homology with the ChaC family proteins that function as γ-glutamyl cyclotransferases, which degrade glutathione (GSH), a tripeptide that plays important roles in the plant immune system. RipAY significantly inhibited yeast growth and simultaneously induced rapid GSH depletion when expressed in yeast cells. The in vitro GSH degradation activity of RipAY is specifically activated by eukaryotic factors in the yeast and plant extracts. Biochemical purification of the yeast protein identified that RipAY is activated by thioredoxin TRX2. On the other hand, RipAY was not activated by bacterial thioredoxins. Interestingly, RipAY was activated by plant h -type thioredoxins that exist in large amounts in the plant cytosol, but not by chloroplastic m -, f -, x -, y - and z -type thioredoxins, in a thiol-independent manner. The transient expression of RipAY decreased the GSH level in plant cells and affected the flg22-triggered production of reactive oxygen species (ROS) and expression of pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) marker genes in Nicotiana benthamiana leaves. These results indicate that RipAY is activated by host cytosolic thioredoxins and degrades GSH specifically in plant cells to suppress plant immunity. IMPORTANCE Ralstonia solanacearum is the causal agent of bacterial wilt disease of plants. This pathogen injects virulence effector proteins into host cells to suppress disease resistance responses of plants. In this article, we report a biochemical activity of R. solanacearum effector protein RipAY. RipAY can degrade GSH, a tripeptide that plays important roles in the plant immune system, with its γ-glutamyl cyclotransferase activity. The high GSH degradation activity of RipAY is considered to be a good weapon for this bacterium to suppress plant immunity. However, GSH also plays important roles in bacterial tolerance to various stresses and growth. Interestingly, RipAY has an excellent safety mechanism to prevent unwanted firing of its enzyme activity in bacterial cells because RipAY is specifically activated by host eukaryotic thioredoxins. This study also reveals a novel host plant protein acting as a molecular switch for effector activation.

scholarly journals Response of immunocompetent and immunosuppressed Spodoptera littoralis larvae to baculovirus infection

2006 ◽  
Vol 87 (8) ◽  
pp. 2217-2225 ◽  
Author(s):  
Hadassah Rivkin ◽  
Jeremy A. Kroemer ◽  
Alexander Bronshtein ◽  
Eduard Belausov ◽  
Bruce A. Webb ◽  
...  
Keyword(s):  
Immune System ◽  
Immune Responses ◽  
Spodoptera Littoralis ◽  
Fluorescent Protein ◽  
Oral Route ◽  
Green Fluorescent Protein Gene ◽  
Successful Infection ◽  
Baculovirus Infection ◽  
Budded Virus ◽  
Green Fluorescent

The Mediterranean lepidopteran pest Spodoptera littoralis is highly resistant to infection with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV) via the oral route, but highly sensitive to infection with budded virus (BV) via the intrahaemocoelic route. To study the fate of AcMNPV infection in S. littoralis, vHSGFP, an AcMNPV recombinant that expresses the reporter green fluorescent protein gene under the control of the Drosophila heat-shock promoter, and high-resolution fluorescence microscopy were utilized. S. littoralis fourth-instar larvae infected orally with vHSGFP showed melanization and encapsulation of virus-infected tracheoblast cells serving the midgut columnar cells. At 72 h post-infection, the viral foci were removed during the moult clearing the infection. Thus, oral infection was restricted by immune responses to the midgut and midgut-associated tracheal cells. By contrast, injection of BV into the haemocoel resulted in successful infection of tracheoblasts, followed by spread of the virus through the tracheal epidermis to other tissues. However, in contrast to fully permissive infections where tracheoblasts and haemocytes are equally susceptible to infection, a severe limitation to vHSGFP infection of haemocytes was observed. To investigate the resistance of S. littoralis haemocytes to BV infection with AcMNPV, the larval immune system was suppressed with the Chelonus inanitus polydnavirus or a putatively immunosuppressive polydnavirus gene, P-vank-1. Both treatments increased the susceptibility of S. littoralis larvae to AcMNPV. It is concluded that the resistance of S. littoralis to AcMNPV infection involves both humoral and cellular immune responses that act at the gut and haemocyte levels. The results also support the hypothesis that tracheolar cells mediate establishment of systemic baculovirus infections in lepidopteran larvae. The finding that polydnaviruses and their encoded genes synergize baculovirus infection also provides an approach to dissecting the responses of the lepidopteran immune system to viruses by using specific polydnavirus immunosuppressive genes.

scholarly journals Structures of Phytophthora RXLR Effector Proteins

2011 ◽  
Vol 286 (41) ◽  
pp. 35834-35842 ◽  
Author(s):  
Laurence S. Boutemy ◽  
Stuart R. F. King ◽  
Joe Win ◽  
Richard K. Hughes ◽  
Thomas A. Clarke ◽  
...  
Keyword(s):  
Immune System ◽  
Molecular Mechanisms ◽  
Tandem Repeats ◽  
Effector Proteins ◽  
Functional Adaptation ◽  
In Planta ◽  
Effector Domains ◽  
The Core ◽  
Plant Immune System ◽  
Molecular Stability

Phytopathogens deliver effector proteins inside host plant cells to promote infection. These proteins can also be sensed by the plant immune system, leading to restriction of pathogen growth. Effector genes can display signatures of positive selection and rapid evolution, presumably a consequence of their co-evolutionary arms race with plants. The molecular mechanisms underlying how effectors evolve to gain new virulence functions and/or evade the plant immune system are poorly understood. Here, we report the crystal structures of the effector domains from two oomycete RXLR proteins, Phytophthora capsici AVR3a11 and Phytophthora infestans PexRD2. Despite sharing <20% sequence identity in their effector domains, they display a conserved core α-helical fold. Bioinformatic analyses suggest that the core fold occurs in ∼44% of annotated Phytophthora RXLR effectors, both as a single domain and in tandem repeats of up to 11 units. Functionally important and polymorphic residues map to the surface of the structures, and PexRD2, but not AVR3a11, oligomerizes in planta. We conclude that the core α-helical fold enables functional adaptation of these fast evolving effectors through (i) insertion/deletions in loop regions between α-helices, (ii) extensions to the N and C termini, (iii) amino acid replacements in surface residues, (iv) tandem domain duplications, and (v) oligomerization. We hypothesize that the molecular stability provided by this core fold, combined with considerable potential for plasticity, underlies the evolution of effectors that maintain their virulence activities while evading recognition by the plant immune system.

scholarly journals Genome-Wide Identification of a Large Repertoire of Ralstonia solanacearum Type III Effector Proteins by a New Functional Screen

2010 ◽  
Vol 23 (3) ◽  
pp. 251-262 ◽  
Author(s):  
Takafumi Mukaihara ◽  
Naoyuki Tamura ◽  
Masaki Iwabuchi
Keyword(s):  
Ralstonia Solanacearum ◽  
Type Iii Secretion ◽  
Plant Cells ◽  
Amino Acid Sequences ◽  
Effector Proteins ◽  
Type Iii ◽  
Functional Screen ◽  
Genome Wide ◽  
Large Repertoire ◽  
Insertion Mutants

The gram-negative plant-pathogenic bacterium Ralstonia solanacearum utilizes the hypersensitive response and pathogenicity (Hrp) type III secretion system (T3SS) to cause disease in plants. To determine the entire repertoire of effector proteins possessed by R. solanacearum RS1000, we constructed a transposon carrying a calmodulin-dependent adenylate cyclase reporter that can be used to specifically detect rip (Ralstonia protein injected into plant cells) genes by monitoring the cAMP level in plant leaves inoculated with insertion mutants. From the new functional screen using this transposon, we identified 38 new Rip proteins translocated into plant cells via the Hrp T3SS. In addition, most of the 34 known effectors of RS1000 could be detected by the screen, except for three effectors that appear to be small in size or only weakly expressed. Finally, we identified 72 Rips in RS1000, which include 68 effector proteins classified into over 50 families and four extracellular components of the Hrp T3SS. Interestingly, one-third of the effectors are specific to R. solanacearum. Many effector proteins contain various repeated amino acid sequences or known enzyme motifs. We also show that most of the R. solanacearum effector proteins, but not Hrp extracellular components, require an Hrp-associated protein, HpaB, for their effective translocation into plant cells.

scholarly journals CaSK23, a Putative GSK3/SHAGGY-Like Kinase of Capsicum annuum, Acts as a Negative Regulator of Pepper’s Response to Ralstonia solanacearum Attack

2018 ◽  
Vol 19 (9) ◽  
pp. 2698 ◽  
Author(s):  
Ailian Qiu ◽  
Ji Wu ◽  
Yufen Lei ◽  
Yiting Cai ◽  
Song Wang ◽  
...  
Keyword(s):  
Ralstonia Solanacearum ◽  
Plant Immunity ◽  
Constitutive Expression ◽  
Negative Regulator ◽  
Virus Induced Gene Silencing ◽  
Pr Genes ◽  
Down Regulation ◽  
Genes Encoding ◽  
Transient Overexpression ◽  
Pepper Plants

GSK3-like kinases have been mainly implicated in the brassinosteroids (BR) pathway and, therefore, in plant growth, development, and responses to abiotic stresses; however, their roles in plant immunity remain poorly understood. Herein, we present evidence that CaSK23, a putative GSK3/SHAGGY-like kinase in pepper, acts as a negative regulator in pepper’s response to Ralstonia solanacearum (R. solanacearum) inoculation (RSI). Data from quantitative RT-PCR (qRT-PCR) showed that the constitutively-expressed CaSK23 in pepper leaves was down-regulated by RSI, as well as by exogenously-applied salicylic acid (SA) or methyl jasomonate (MeJA). Silencing of CaSK23 by virus-induced gene silencing (VIGS) decreased the susceptibility of pepper plants to RSI, coupled with up-regulation of the tested genes encoding SA-, JA-, and ethylene (ET)-dependent pathogenesis-related (PR) proteins. In contrast, ectopic overexpression (OE) of CaSK23 conferred a compromised resistance of tobacco plants to RSI, accompanied by down-regulation of the tested immunity-associated SA-, JA-, and ET-dependent PR genes. In addition, transient overexpression of CaSK23 in pepper plants consistently led to down-regulation of the tested SA-, JA-, and ET-dependent PR genes. We speculate that CaSK23 acts as a negative regulator in pepper immunity and its constitutive expression represses pepper immunity in the absence of pathogens. On the other hand, its decreased expression derepresses immunity when pepper plants are attacked by pathogens.

Isolation of Ralstonia solanacearum hrpB constitutive mutants and secretion analysis of hrpB-regulated gene products that share homology with known type III effectors and enzymes

Microbiology ◽  
2005 ◽  
Vol 151 (9) ◽  
pp. 2873-2884 ◽  
Author(s):  
Naoyuki Tamura ◽  
Yukio Murata ◽  
Takafumi Mukaihara
Keyword(s):  
Pseudomonas Syringae ◽  
Ralstonia Solanacearum ◽  
Type Iii Secretion ◽  
Effector Proteins ◽  
Gene Products ◽  
Nudix Hydrolase ◽  
Type Iii Effectors ◽  
Type Iii ◽  
Extracellular Secretion ◽  
Culture Supernatants

The Hrp type III secretion system (TTSS) is essential for the pathogenicity of the Gram-negative plant pathogen Ralstonia solanacearum. To examine the secretion of type III effector proteins via the Hrp TTSS, a screen was done of mutants constitutively expressing the hrpB gene, which encodes an AraC-type transcriptional activator for the hrp regulon. A mutant was isolated that in an hrp-inducing medium expresses several hrpB-regulated genes 4·9–83-fold higher than the wild-type. R. solanacearum Hrp-secreted outer proteins PopA and PopC were secreted at high levels into the culture supernatants of the hrpB constitutive (hrpB c) mutant. Using hrpB c mutants, the extracellular secretion of several hrpB-regulated (hpx) gene products that share homology with known type III effectors and enzymes was examined. Hpx23, Hpx24 and Hpx25, which are similar in sequence to Pseudomonas syringae pv. tomato effector proteins HopPtoA1, HolPtoR and HopPtoD1, are also secreted via the Hrp TTSS in R. solanacearum. The secretion of two hpx gene products that share homology with known enzymes, glyoxalase I (Hpx19) and Nudix hydrolase (Hpx26), was also examined. Hpx19 is accumulated inside the cell, but interestingly, Hpx26 is secreted outside the cell as an Hrp-secreted outer protein, suggesting that Hpx19 functions intracellularly but Hpx26 is a novel effector protein of R. solanacearum.

scholarly journals Activation of TIR signaling is required for pattern-triggered immunity

2020 ◽  
Author(s):  
Hainan Tian ◽  
Siyu Chen ◽  
Zhongshou Wu ◽  
Kevin Ao ◽  
Hoda Yaghmaiean ◽  
...  
Keyword(s):  
Immune Responses ◽  
Interleukin 1 ◽  
Defense Responses ◽  
Effector Proteins ◽  
Leucine Rich Repeat ◽  
Number Of Genes ◽  
Genes Encoding ◽  
Signaling Activation ◽  
Tir Domains ◽  
The Relationship

AbstractPlant immune responses are mainly activated by two types of receptors. Plasma membrane-localized pattern recognition receptors (PRRs) recognize conserved features of microbes, and intracellular nucleotide-binding leucine rich repeat receptors (NLRs) recognize effector proteins from pathogens. NLRs possessing N-terminal Toll/interleukin-1 receptor (TIR) domains (TNLs) activate two parallel signaling pathways via the EDS1/PAD4/ADR1s and the EDS1/SAG101/NRG1s modules. The relationship between PRR-mediated pattern-triggered immunity (PTI) and TIR signaling is unclear. Here we report that activation of TIR signaling plays a key role in PTI. Blocking TIR signaling by knocking out components of the EDS1/PAD4/ADR1s and EDS1/SAG101/NRG1s modules results in attenuated PTI responses such as reduced salicylic acid (SA) levels and expression of defense genes, and compromised resistance against pathogens. Consistently, PTI is attenuated in transgenic plants that have reduced accumulation of NLRs. Upon treatment with PTI elicitors such as flg22 and nlp20, a large number of genes encoding TNLs or TIR domain-containing proteins are rapidly induced, likely responsible for activating TIR signaling during PTI. In support, overexpression of some of these genes results in activation of defense responses. Overall, our study reveals that TIR signaling activation is an important mechanism for boosting plant defense during PTI.

scholarly journals The bacterial type III-secreted protein AvrRps4 is a bipartite effector

2017 ◽  
Author(s):  
◽  
Morgan K. Halane
Keyword(s):  
Cell Death ◽  
Immune System ◽  
Resistance Genes ◽  
Pathogenic Bacteria ◽  
Global Climate ◽  
Plant Cells ◽  
Effector Proteins ◽  
Effector Domain ◽  
Cell Death Response ◽  
The Face

Like humans, plants have an immune system to protect themselves against invading pathogens. Unlike humans, however, the plant immune system is inborn and genetically predetermined by resistance genes. Pathogenic bacteria secrete proteins (effectors) into plant cells which manipulate the host cell, often to the benefit of the pathogen. The proteins encoded by resistance genes in resistant plants can specifically detect these effectors and ramp up a potent immune response, often resulting in cell death. By studying these effector proteins and how hosts can recognize them we hope to generate novel, durable methods to protect economically important plants from devastating pathogens. With a rapidly growing population in the face of global climate change it is more important than ever to protect the plants which we all use for food, fiber, and fuel. The effector AvrRps4 is recognized by the protein pair RPS4/RRS1. After delivery into plant cells AvrRps4 is processed into two parts (AvrRps4N and AvrRps4C). AvrRps4C was shown to trigger a cell death response in turnip and has been the most well-studied domain of AvrRps4. My research shows that AvrRps4N is also a functional effector domain. In some plants, in the absence of AvrRps4C, it enhances bacterial virulence. In resistant plants it enhances immunity in the presence of AvrRps4C. Finally, I show that AvrRps4N alone can trigger cell death on some plants, further confirming its role as a bona fide effector domain.

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

2019 ◽  
Vol 32 (8) ◽  
pp. 986-1000 ◽  
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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