Two Distinct Pseudomonas Effector Proteins Interact with the Pto Kinase and Activate Plant Immunity

Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines translocating effector proteins into the cytoplasm of eukaryotic cells. They have been intensively studied for their important roles in animal and plant bacterial diseases. Over the past two decades, genome sequencing has unveiled their ubiquitous distribution in many taxa of Gram-negative bacteria, including plant-beneficial ones. Here, we discuss the distribution and functions of the T3SS in two agronomically important bacterial groups: the symbiotic nodule-forming nitrogen-fixing rhizobia and the free-living plant-beneficial Pseudomonas spp. In legume-rhizobia symbiosis, T3SSs and their cognate effectors play important roles, including the modulation of the plant immune response and the initiation of the nodulation process in some cases. In plant-beneficial Pseudomonas spp., the roles of T3SSs are not fully understood, but pertain to plant immunity suppression, biocontrol against eukaryotic plant pathogens, mycorrhization facilitation, and possibly resistance against protist predation. The diversity of T3SSs in plant-beneficial bacteria points to their important roles in multifarious interkingdom interactions in the rhizosphere. We argue that the gap in research on T3SSs in plant-beneficial bacteria must be bridged to better understand bacteria/eukaryotes rhizosphere interactions and to support the development of efficient plant-growth promoting microbial inoculants.

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Three LysM effectors of Zymoseptoria tritici collectively disarm chitin-triggered plant immunity

10.1101/2020.06.24.169789 ◽

2020 ◽

Cited By ~ 1

Author(s):

Hui Tian ◽

Craig I. MacKenzie ◽

Luis Rodriguez-Moreno ◽

Grardy C.M. van den Berg ◽

Hongxin Chen ◽

...

Keyword(s):

Plant Immunity ◽

Effector Proteins ◽

Septoria Tritici Blotch ◽

Septoria Tritici ◽

Zymoseptoria Tritici ◽

Plant Host ◽

Fungal Cell ◽

Molecular Pattern ◽

Fungal Hyphae ◽

Ros Burst

SUMMARYChitin is a major structural component of fungal cell walls and acts as a microbe-associated molecular pattern (MAMP) that, upon recognition by a plant host, triggers the activation of immune responses. In order to avoid the activation of these responses, the Septoria tritici blotch (STB) pathogen of wheat, Zymoseptoria tritici, secretes LysM effector proteins. Previously, the LysM effectors Mg1LysM and Mg3LysM were shown to protect fungal hyphae against host chitinases. Furthermore, Mg3LysM, but not Mg1LysM, was shown to suppress chitin-induced reactive oxygen species (ROS) production. Whereas initially a third LysM effector gene was disregarded as a presumed pseudogene, we now provide functional data to show that also this gene encodes a LysM effector, named Mgx1LysM, that is functional during wheat colonization. While Mg3LysM confers a major contribution to Z. tritici virulence, Mgx1LysM and Mg1LysM contribute to Z. tritici virulence with smaller effects. All three LysM effectors display partial functional redundancy. We furthermore demonstrate that Mgx1LysM binds chitin, suppresses the chitin-induced ROS burst and is able to protect fungal hyphae against chitinase hydrolysis. Finally, we demonstrate that Mgx1LysM is able to undergo chitin-induced polymerisation. Collectively, our data show that Zymoseptoria tritici utilizes three LysM effectors to disarm chitin-triggered wheat immunity.

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Downy Mildew effector HaRxL21 interacts with the transcriptional repressor TOPLESS to promote pathogen susceptibility

10.1101/2020.04.29.066688 ◽

2020 ◽

Author(s):

Sarah Harvey ◽

Priyanka Kumari ◽

Dmitry Lapin ◽

Thomas Griebel ◽

Richard Hickman ◽

...

Keyword(s):

Downy Mildew ◽

Transcriptional Repression ◽

Plant Immunity ◽

Effector Proteins ◽

Host Susceptibility ◽

Close Relative ◽

C Terminus ◽

Rxlr Effector ◽

Oomycete Pathogen ◽

Hyaloperonospora Arabidopsidis

AbstractHyaloperonospora arabidopsidis (Hpa) is an oomycete pathogen causing Arabidopsis downy mildew. Effector proteins secreted from the pathogen into the plant play key roles in promoting infection by suppressing plant immunity and manipulating the host to the pathogen’s advantage. One class of oomycete effectors share a conserved ‘RxLR’ motif critical for their translocation into the host cell. Here we characterize the interaction between an RxLR effector, HaRxL21 (RxL21), and the Arabidopsis transcriptional co-repressor Topless (TPL). We establish that RxL21 and TPL interact via an EAR motif at the C-terminus of the effector, mimicking the host plant mechanism for recruiting TPL to sites of transcriptional repression. We show that this motif, and hence interaction with TPL, is necessary for the virulence function of the effector. Furthermore, we provide evidence that RxL21 uses the interaction with TPL, and its close relative TPL-related 1, to repress plant immunity and enhance host susceptibility to both biotrophic and necrotrophic pathogens.

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Molecular Evolution ofPseudomonas syringaeType III Secreted Effector Proteins

10.1101/503219 ◽

2018 ◽

Author(s):

Marcus M. Dillon ◽

Renan N.D. Almeida ◽

Bradley Laflamme ◽

Alexandre Martel ◽

Bevan S. Weir ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Global Analysis ◽

Plant Immunity ◽

Amino Acid Level ◽

Effector Proteins ◽

Gene Gain ◽

Host Immune System ◽

Virulence Potential ◽

History Of ◽

Complex Variation

ABSTRACTDiverse Gram-negative pathogens likePseudomonas syringaeemploy type III secreted effector (T3SE) proteins as primary virulence factors that combat host immunity and promote disease. T3SEs can also be recognized by plant hosts and activate an effector triggered immune (ETI) response that shifts the interaction back towards plant immunity. Consequently, T3SEs are pivotal in determining the virulence potential of individualP. syringaestrains, and ultimately restrictP. syringaepathogens to a subset of potential hosts that are unable to recognize their repertoires of T3SEs. While a number of effector families are known to be present in theP. syringaespecies complex, one of the most persistent challenges has been documenting the complex variation in T3SE contents across a diverse collection of strains. Using the entire pan-genome of 494P. syringaestrains isolated from more than 100 hosts, we conducted a global analysis of all known and putative T3SEs. We identified a total of 14,613 T3SEs, 4,636 of which were unique at the amino acid level, and show that T3SE repertoires of differentP. syringaestrains vary dramatically, even among strains isolated from the same hosts. We also find that dramatic diversification has occurred within many T3SE families, and in many cases find strong signatures of positive selection. Furthermore, we identify multiple gene gain and loss events for several families, demonstrating an important role of horizontal gene transfer (HGT) in the evolution ofP. syringaeT3SEs. These analyses provide insight into the evolutionary history ofP. syringaeT3SEs as they co-evolve with the host immune system, and dramatically expand the database ofP. syringaeT3SEs alleles.

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A Phytophthora sojae CRN effector mediates phosphorylation and degradation of plant aquaporin proteins to suppress host immune signaling

PLoS Pathogens ◽

10.1371/journal.ppat.1009388 ◽

2021 ◽

Vol 17 (3) ◽

pp. e1009388

Author(s):

Gan Ai ◽

Qingyue Xia ◽

Tianqiao Song ◽

Tianli Li ◽

Hai Zhu ◽

...

Keyword(s):

Higher Plants ◽

Plant Immunity ◽

Phytophthora Capsici ◽

Kinase Domain ◽

Phytophthora Sojae ◽

Effector Proteins ◽

Ros Accumulation ◽

Oomycete Pathogen ◽

Cellular Defenses

Phytophthora genomes encode a myriad of Crinkler (CRN) effectors, some of which contain putative kinase domains. Little is known about the host targets of these kinase-domain-containing CRNs and their infection-promoting mechanisms. Here, we report the host target and functional mechanism of a conserved kinase CRN effector named CRN78 in a notorious oomycete pathogen, Phytophthora sojae. CRN78 promotes Phytophthora capsici infection in Nicotiana benthamiana and enhances P. sojae virulence on the host plant Glycine max by inhibiting plant H2O2 accumulation and immunity-related gene expression. Further investigation reveals that CRN78 interacts with PIP2-family aquaporin proteins including NbPIP2;2 from N. benthamiana and GmPIP2-13 from soybean on the plant plasma membrane, and membrane localization is necessary for virulence of CRN78. Next, CRN78 promotes phosphorylation of NbPIP2;2 or GmPIP2-13 using its kinase domain in vivo, leading to their subsequent protein degradation in a 26S-dependent pathway. Our data also demonstrates that NbPIP2;2 acts as a H2O2 transporter to positively regulate plant immunity and reactive oxygen species (ROS) accumulation. Phylogenetic analysis suggests that the phosphorylation sites of PIP2 proteins and the kinase domains of CRN78 homologs are highly conserved among higher plants and oomycete pathogens, respectively. Therefore, this study elucidates a conserved and novel pathway used by effector proteins to inhibit host cellular defenses by targeting and hijacking phosphorylation of plant aquaporin proteins.

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Recent advances in the regulation of plant immunity by S-nitrosylation

Journal of Experimental Botany ◽

10.1093/jxb/eraa454 ◽

2020 ◽

Author(s):

Jibril Lubega ◽

Saima Umbreen ◽

Gary J Loake

Keyword(s):

Plant Immunity ◽

Protein S ◽

Cellular Protein ◽

Effector Proteins ◽

Host Cells ◽

Immune Functions ◽

Post Translational Modification ◽

No Donor ◽

Reactive Protein ◽

Recent Advances

Abstract S-nitrosylation, the addition of a nitric oxide (NO) moiety to a reactive protein cysteine (Cys) thiol, to form a protein S-nitrosothiol (SNO), is emerging as a key regulatory post-translational modification (PTM) to control the plant immune response. NO also S-nitrosylates the antioxidant tripeptide, glutathione, to form S-nitrosoglutathione (GSNO), both a storage reservoir of NO bioactivity and a natural NO donor. GSNO and, by extension, S-nitrosylation, are controlled by GSNO reductase1 (GSNOR1). The emerging data suggest that GSNOR1 itself is a target of NO-mediated S-nitrosylation, which subsequently controls its selective autophagy, regulating cellular protein SNO levels. Recent findings also suggest that S-nitrosylation may be deployed by pathogen-challenged host cells to counteract the effect of delivered microbial effector proteins that promote pathogenesis and by the pathogens themselves to augment virulence. Significantly, it also appears that S-nitrosylation may regulate plant immune functions by controlling SUMOylation, a peptide-based PTM. In this context, global SUMOylation is regulated by S-nitrosylation of SUMO conjugating enzyme 1 (SCE1) at Cys139. This redox-based PTM has also been shown to control the function of a key zinc finger transcriptional regulator during the establishment of plant immunity. Here, we provide an update of these recent advances.

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Regulation of Tomato Prf by Pto-like Protein Kinases

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-4-0391 ◽

2009 ◽

Vol 22 (4) ◽

pp. 391-401 ◽

Cited By ~ 36

Author(s):

Tatiana S. Mucyn ◽

Ai-Jiuan Wu ◽

Alexi L. Balmuth ◽

Julia Maryam Arasteh ◽

John P. Rathjen

Keyword(s):

Protein Kinases ◽

Pseudomonas Syringae ◽

Kinase Activity ◽

Negative Regulation ◽

Effector Proteins ◽

Leucine Rich Repeats ◽

Direct Recognition ◽

Fen Kinase ◽

Bacterial Effectors ◽

Pto Kinase

Tomato Prf encodes a nucleotide-binding domain shared by Apaf-1, certain R proteins, and CED-4 fused to C-terminal leucine-rich repeats (NBARC-LRR) protein that is required for bacterial immunity to Pseudomonas syringae and sensitivity to the organophosphate fenthion. The signaling pathways involve two highly related protein kinases. Pto kinase mediates direct recognition of the bacterial effector proteins AvrPto or AvrPtoB. Fen kinase is required for fenthion sensitivity and recognition of bacterial effectors related to AvrPtoB. The role of Pto and its association with Prf has been characterized but Fen is poorly described. We show that, similar to Pto, Fen requires N-myristoylation and kinase activity for signaling and interacts with the N-terminal domain of Prf. Thus, the mechanisms of activation of Prf by the respective protein kinases are similar. Prf–Fen interaction is underlined by coregulatory mechanisms in which Prf negatively regulates Fen, most likely by controlling kinase activity. We further characterized negative regulation of Prf by Pto, and show that regulation is mediated by the previously described negative regulatory patch. Remarkably, the effectors released negative regulation of Prf in a manner dependent on Pto kinase activity. The data suggest a model in which Prf associates generally with Pto-like kinases in tightly regulated complexes, which are activated by effector-mediated disruption of negative regulation. Release of negative regulation may be a general feature of activation of NBARC-LRR proteins by cognate effectors.

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Identification and Functional Analysis of AopN, an Acidovorax Citrulli Effector that Induces Programmed Cell Death in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms21176050 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6050 ◽

Cited By ~ 1

Author(s):

Xiaoxiao Zhang ◽

Mei Zhao ◽

Jie Jiang ◽

Linlin Yang ◽

Yuwen Yang ◽

...

Keyword(s):

Functional Analysis ◽

Cell Death ◽

Programmed Cell Death ◽

Pathogenic Bacteria ◽

Plant Immunity ◽

Effector Proteins ◽

Economic Losses ◽

Homologous Protein ◽

Acidovorax Citrulli ◽

Effector Triggered Immunity

Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, seriously affects watermelon and other cucurbit crops, resulting in significant economic losses. However, the pathogenicity mechanism of A. citrulli is not well understood. Plant pathogenic bacteria often suppress the plant immune response by secreting effector proteins. Thus, identifying A. citrulli effector proteins and determining their functions may improve our understanding of the underlying pathogenetic mechanisms. In this study, a novel effector, AopN, which is localized on the cell membrane of Nicotiana benthamiana, was identified. The functional analysis revealed that AopN significantly inhibited the flg22-induced reactive oxygen species burst. AopN induced a programmed cell death (PCD) response. Unlike its homologous protein, the ability of AopN to induce PCD was dependent on two motifs of unknown functions (including DUP4129 and Cpta_toxin), but was not dependent on LXXLL domain. More importantly, the virulence of the aopN mutant of A. citrulli in N. benthamiana significantly decreased, indicating that it was a core effector. Further analysis revealed that AopN interacted with watermelon ClHIPP and ClLTP, which responds to A. citrulli strain Aac5 infection at the transcription level. Collectively, these findings indicate that AopN suppresses plant immunity and activates the effector-triggered immunity pathway.

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The type III effector HopF2Pto targets Arabidopsis RIN4 protein to promote Pseudomonas syringae virulence

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0904739107 ◽

2010 ◽

Vol 107 (5) ◽

pp. 2349-2354 ◽

Cited By ~ 92

Author(s):

Mike Wilton ◽

Rajagopal Subramaniam ◽

James Elmore ◽

Corinna Felsensteiner ◽

Gitta Coaker ◽

...

Keyword(s):

Pseudomonas Syringae ◽

Plant Immunity ◽

Effector Proteins ◽

Type Iii ◽

Pathogen Effector ◽

Effector Triggered Immunity ◽

Type Iii Secretion Effector ◽

Virulence Target

Plant immunity can be induced by two major classes of pathogen-associated molecules. Pathogen- or microbe-associated molecular patterns (PAMPs or MAMPs) are conserved molecular components of microbes that serve as “non-self” features to induce PAMP-triggered immunity (PTI). Pathogen effector proteins used to promote virulence can also be recognized as “non-self” features or induce a “modified-self” state that can induce effector-triggered immunity (ETI). The Arabidopsis protein RIN4 plays an important role in both branches of plant immunity. Three unrelated type III secretion effector (TTSE) proteins from the phytopathogen Pseudomonas syringae, AvrRpm1, AvrRpt2, and AvrB, target RIN4, resulting in ETI that effectively restricts pathogen growth. However, no pathogenic advantage has been demonstrated for RIN4 manipulation by these TTSEs. Here, we show that the TTSE HopF2Pto also targets Arabidopsis RIN4. Transgenic plants conditionally expressing HopF2Pto were compromised for AvrRpt2-induced RIN4 modification and associated ETI. HopF2Pto interfered with AvrRpt2-induced RIN4 modification in vitro but not with AvrRpt2 activation, suggestive of RIN4 targeting by HopF2Pto. In support of this hypothesis, HopF2Pto interacted with RIN4 in vitro and in vivo. Unlike AvrRpm1, AvrRpt2, and AvrB, HopF2Pto did not induce ETI and instead promoted P. syringae growth in Arabidopsis. This virulence activity was not observed in plants genetically lacking RIN4. These data provide evidence that RIN4 is a major virulence target of HopF2Pto and that a pathogenic advantage can be conveyed by TTSEs that target RIN4.

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Bacterial Effector HopF2 Suppresses Arabidopsis Innate Immunity at the Plasma Membrane

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-07-10-0150 ◽

2011 ◽

Vol 24 (5) ◽

pp. 585-593 ◽

Cited By ~ 39

Author(s):

Shujing Wu ◽

Dongping Lu ◽

Mehdi Kabbage ◽

Hai-Lei Wei ◽

Bryan Swingle ◽

...

Keyword(s):

Innate Immunity ◽

Plasma Membrane ◽

Pseudomonas Syringae ◽

Plant Immunity ◽

Mapk Signaling ◽

Effector Proteins ◽

Host Cells ◽

Mitogen Activated Protein Kinase ◽

Immune Response Gene ◽

Secretion Systems

Many bacterial pathogens inject a cocktail of effector proteins into host cells through type III secretion systems. These effectors act in concert to modulate host physiology and immune signaling, thereby promoting pathogenicity. In a search for additional Pseudomonas syringae effectors in suppressing plant innate immunity triggered by pathogen or microbe-associated molecular patterns (PAMPs or MAMPs), we identified P. syringae tomato DC3000 effector HopF2 as a potent suppressor of early immune-response gene transcription and mitogen-activated protein kinase (MAPK) signaling activated by multiple MAMPs, including bacterial flagellin, elongation factor Tu, peptidoglycan, lipopolysaccharide and HrpZ1 harpin, and fungal chitin. The conserved surface-exposed residues of HopF2 are essential for its MAMP suppression activity. HopF2 is targeted to the plant plasma membrane through a putative myristoylation site, and the membrane association appears to be required for its MAMP-suppression function. Expression of HopF2 in plants potently diminished the flagellin-induced phosphorylation of BIK1, a plasma membrane–associated cytoplasmic kinase that is rapidly phosphorylated within one minute upon flagellin perception. Thus, HopF2 likely intercepts MAMP signaling at the plasma membrane immediately of signal perception. Consistent with the potent suppression function of multiple MAMP signaling, expression of HopF2 in transgenic plants compromised plant nonhost immunity to bacteria P. syringae pv. Phaseolicola and plant immunity to the necrotrophic fungal pathogen Botrytis cinerea.

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