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Special Feature Articles: Parlez-vous effectors? Francis Martin

Host-selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis–wheat interaction Lynda M. Ciuffetti, Viola A. Manning, Iovanna Pandelova, Melania Figueroa Betts and J. Patrick Martinez

Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria Robert Szczesny et al.

Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins Jianying Wang et al.

Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum Stéphane Genin

Towards population genomics of effector–effector target interactions Ryohei Terauchi and Kentaro Yoshida

Specific resistances against Pseudomonas syringae effectors AvrB and AvrRpm1 have evolved differently in common bean (Phaseolus vulgaris), soybean (Glycine max), and Arabidopsis thaliana Nicolas W. G. Chen et al.

The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis

The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity Alberto P. Macho, Carlos M. Guevara, Pablo Tornero, Javier RuizAlbert and Carmen R. Beuzón

Intracellular expression of a host-selective toxin, ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1 Viola A. Manning, Ashley L. Chu, Steven R. Scofield and Lynda M. Ciuffetti

Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae Patrick Römer et al.

Yuliya Khrunyk, Karin Münch, Kerstin Schipper, Andrei N. Lupas and Regine Kahmann

Suppression of the AvrBs1-specific hypersensitive response by the YopJ effector homolog AvrBsT from Xanthomonas depends on a SNF1-related kinase

Two virulence determinants of type III effector AvrPto are functionally conserved in diverse Pseudomonas syringae pathovars

Robert Szczesny et al.

Hanh P. Nguyen, Inhwa Yeam, Aurelie Angot and Gregory B. Martin

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Introduction Effectors are defined as molecules that manipulate host-cell structure and function, thereby facilitating infection (virulence factors or toxins) and/or triggering defense responses (avirulence factors or elicitors). This dual (and conflicting) activity of effectors has been broadly reported in many plant–microbial pathosystems and is proving to be a rapidly developing field. Research in this area currently employs a variety of approaches (biochemical, physiological and developmental), together with cellular biology, bioinformatics, functional genomics and proteomics that is quickly moving towards genome -wide and evolutionary analyses. The scene is thus set for us to start unpicking how molecular information is exchanged among species, and how this information is related to the functional character of the plant and microbial interface. In addition to topical reviews, this New Phytologist Feature (published in issue 187-4, 2010) presents some exciting results regarding the evolution, trafficking and targeting of effectors.


Research review Host-selective toxins, Ptr ToxA and Ptr ToxB, as necrotrophic effectors in the Pyrenophora tritici-repentis–wheat interaction Author for correspondence: Lynda M. Ciuffetti Tel: +1 541 737 2188 Email: ciuffetL@science.oregonstate.edu

Lynda M. Ciuffetti, Viola A. Manning, Iovanna Pandelova, Melania Figueroa Betts and J. Patrick Martinez

Summary New Phytologist (2010) 187: 911–919 doi: 10.1111/j.1469-8137.2010.03362.x

Keywords: effector; host-selective toxins; inverse gene-for-gene; Ptr ToxA; Ptr ToxB; Pyrenophora tritici-repentis; susceptibility; tan spot of wheat

Host-selective toxins (HSTs) are effectors produced by some necrotrophic pathogenic fungi that typically confer the ability to cause disease. Often, diseases caused by pathogens that produce HSTs follow an inverse gene-for-gene model where toxin production is required for the ability to cause disease and a single locus in the host is responsible for toxin sensitivity and disease susceptibility. Pyrenophora tritici-repentis represents an ideal pathogen for studying the biological significance of such inverse gene-for-gene interactions, because it displays a complex race structure based on its production of multiple HSTs. Ptr ToxA and Ptr ToxB are two proteinaceous HSTs produced by P. tritici-repentis that are structurally unrelated and appear to evoke different host responses, yet each toxin confers the ability to cause disease. This review will summarize the current knowledge of how these two dissimilar HSTs display distinct modes of action, yet each confers pathogenicity to P. tritici-repentis.


Research review Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum Author for correspondence: Ste´phane Genin Tel: +33 561285416 Email: sgenin@toulouse.inra.fr

Stéphane Genin

Summary New Phytologist (2010) 187: 920–928 doi: 10.1111/j.1469-8137.2010.03397.x

Keywords: bacterial wilt, infection, pathogenicity, plant resistance, symbiosis.

Ralstonia solanacearum is regarded as one of the world’s most important bacterial plant pathogens because of its aggressiveness, large host range, broad geographical distribution and long persistence in soil and water environments. This root pathogen is an attractive model to investigate the question of host adaptation as it exhibits a remarkably broad host range, being able to infect numerous plant species belonging to different botanical families. Several effector proteins transiting through the type III secretion system have been shown to restrict or extend specifically the host range of the bacterium. Recent investigations on the mechanisms that coordinate changes in gene expression during the passage between saprophytism and life within host tissues have allowed the identification of other molecular determinants implicated in the adaptation of R. solanacearum to its hosts and pathogenesis. Among these determinants are genes involved in chemotaxis, secondary metabolic pathways and the detoxification of various antimicrobial compounds, and genes directing the biosynthesis of phytohormones or adherence factors. The regulation of many of these genes is coordinated by the master pathogenicity regulator HrpG. These hrpG-dependent genes control major steps during the interaction with plant cells, and probably determine the ecological behaviour of the microorganism, being required for the establishment of pathogenesis or mutualism.


Research review Molecular traits controlling host range and adaptation to plants in Ralstonia solanacearum Author for correspondence: R. Terauchi Tel: +81 197 682911 Email: terauchi@ibrc.or.jp

Ryohei Terauchi and Kentaro Yoshida

Summary New Phytologist (2010) 187: 929–939 doi: 10.1111/j.1469-8137.2010.03408.x

Keywords: DNA polymorphism, Magnaporthe oryzae, natural selection, population genomics, rice.

Pathogen–plant host coevolutionary interactions exert strong natural selection on both organisms, specifically on the genes coding for effectors (pathogens), as well as on those coding for effector targets and R proteins (plant hosts). Natural selection leaves behind DNA sequence signatures on such genes and on linked genomic regions. These signatures can readily be detected by studying the patterns of intraspecies polymorphisms and interspecies divergence of the DNA sequences. Recent developments in DNA sequencing technology have made whole-genome studies on patterns of DNA polymorphisms : divergence possible. This type of analysis, called ‘population genomics’, appears to be powerful enough to identify novel effector–effector target genes. Here, we provide an overview of the statistical tools used for population genomics and their applications. This is followed by a brief review of evolutionary studies on plant genes involved in host–pathogen interactions. Finally we provide an example from our study on Magnaporthe oryzae.


Specific resistances against Pseudomonas syringae effectors AvrB and AvrRpm1 have evolved differently in common bean (Phaseolus vulgaris), soybean (Glycine max), and Arabidopsis thaliana Nicolas W. G. Chen, Mireille Sévignac, Vincent Thareau, Ghislaine Magdelenat, Perrine David, Tom Ashfield, Roger W. Innes and Valérie Geffroy

Summary Author for correspondence: Vale´ rie Geffroy Tel: +33 1 69 15 33 65 Email: valerie.geffroy@u-psud.fr

New Phytologist (2010) 187: 941–956 doi: 10.1111/j.1469-8137.2010.03337.x

KEYWORDS: AvrB, AvrRpm1, Phaseolus vulgaris (common bean), Pseudomonas syringae pv phaseolicola, resistance gene evolution.

• In plants, the evolution of specific resistance is poorly understood. Pseudomonas syringae effectors AvrB and AvrRpm1 are recognized by phylogenetically distinct resistance (R) proteins in Arabidopsis thaliana (Brassicaceae) and soybean (Glycine max, Fabaceae). In soybean, these resistances are encoded by two tightly linked R genes, Rpg1-b and Rpg1-r. To study the evolution of these specific resistances, we investigated AvrB- and AvrRpm1-induced responses in common bean (Phaseolus vulgaris, Fabaceae). • Common bean genotypes of various geographical origins were inoculated with P. syringae strains expressing AvrB or AvrRpm1. A common bean recombinant inbred line (RIL) population was used to map R genes to AvrRpm1. • No common bean genotypes recognized AvrB. By contrast, multiple genotypes responded to AvrRpm1, and two independent R genes conferring AvrRpm1specific resistance were mapped to the ends of linkage group B11 (Rpsar-1, for resistance to Pseudomonas syringae effector AvrRpm1 number 1) and B8 (Rpsar-2). Rpsar-1 is located in a region syntenic with the soybean Rpg1 cluster. However, mapping of specific Rpg1 homologous genes suggests that AvrRpm1 recognition evolved independently in common bean and soybean. • The conservation of the genomic position of AvrRpm1-specific genes between soybean and common bean suggests a model whereby specific clusters of R genes are predisposed to evolve recognition of the same effector molecules.


The use of FLP-mediated recombination for the functional analysis of an effector gene family in the biotrophic smut fungus Ustilago maydis Yuliya Khrunyk, Karin Münch, Kerstin Schipper, Andrei N. Lupas and Regine Kahmann

Summary Author for correspondence: R. Kahmann Tel: +49 6421 178 501 Email: kahmann@mpi-marburg.mpg.de

• In the Ustilago maydis genome, several novel secreted effector proteins are encoded by gene families. Because of the limited number of selectable markers, the ability to carry out sequential gene deletions has limited the analysis of effector gene families that may have redundant functions.

New Phytologist (2010) 187: 957–968 doi: 10.1111/j.1469-8137.2010.03413.x

• Here, we established an inducible FLP-mediated recombination system in U. maydis that allows repeated rounds of gene deletion using a single selectable marker (HygR). To avoid genome rearrangements via FRT sites remaining in the genome after excision, different mutated FRT sites were introduced.

KEYWORDS: FLP-mediated recombination, gene family, marker deletion, secreted effector, virulence.

• The FLP-mediated selectable marker-removal technique was successfully applied to delete a family of 11 effector genes (eff1) using five sequential rounds of recombination. We showed that expression of all 11 genes is up-regulated during the biotrophic phase. Strains carrying deletions of 9 or all 11 genes showed a significant reduction in virulence, and this phenotype could be partially complemented by the introduction of differentmembers from the gene family, demonstrating redundancy. • The establishment of the FLP ⁄ FRT system in a plant pathogenic fungus paves the way for analyzing multigene families with redundant functions.


Two virulence determinants of type III effector AvrPto are functionally conserved in diverse Pseudomonas syringae pathovars Hanh P. Nguyen, Inhwa Yeam, Aurelie Angot and Gregory B. Martin

Summary Author for correspondence: Gregory B. Martin Tel: +1 607 2541208 Email: gbm7@cornell.edu

New Phytologist (2010) 187: 969–982 doi: 10.1111/j.1469-8137.2009.03175.x

KEYWORDS: PAMP-triggered immunity, Prf, Pseudomonas syringae, Pto, tobacco, tomato.

• The Pseudomonas syringae pv. tomato type III effector protein AvrPto has two functional domains that contribute additively to its ability to promote pathogen virulence in susceptible tomato plants and also defense responses in resistant tomato and tobacco genotypes. Here, we test the hypothesis that key amino acid residues in these two domains will be conserved even in sequence-divergent AvrPto proteins expressed by diverse P. syringae pathovars. • We cloned avrPto homologs from diverse P. syringae pathovars and characterized the four most diverse homologs from P. syringae pathovars mori, lachrymans, myricae and oryzae for their virulence activity and ability to elicit resistance in tomato and tobacco. • Key residues within the two AvrPto domains are conserved in three of the four homologs and are required for virulence activity and defense elicitation. AvrPtooryzae, lacks conserved residues in each domain, but was found to be recognized by a previously unknown resistance gene in both tomato and tobacco. • Our results indicate that the two virulence domains of AvrPto are conserved in diverse pathovars despite the fact these domains are recognized by certain plant species. AvrPto may therefore function in pathovars infecting diverse plant species by targeting conserved host processes.


Functional characterization of the Xcs and Xps type II secretion systems from the plant pathogenic bacterium Xanthomonas campestris pv vesicatoria Robert Szczesny, Matthias Jordan, Claudia Schramm, Steve Schulz, Virginie Cogez, Ulla Bonas and Daniela Büttner

Summary Author for correspondence: Daniela Büttner Tel: +49 345 5526293 Email: daniela.buettner@genetik.unihalle. de New Phytologist (2010) 187: 983–1002 doi: 10.1111/j.1469-8137.2010.03312.x

KEYWORDS: amylase, bacterial spot disease, cellulase, HrpG, pepper, protease, tomato, type II secretion.

• Type II secretion (T2S) systems of many plant-pathogenic bacteria often secrete cell wall-degrading enzymes into the plant apoplast. • Here, we show that the Xps-T2S system from the plant pathogen Xanthomonas campestris pv vesicatoria (Xcv) promotes disease and contributes to the translocation of effector proteins that are delivered into the plant cell by the type III secretion (T3S) system. • The Xcs-T2S system instead lacks an obvious virulence function. However, individual xcs genes can partially complement mutants in homologous xps genes, indicating that they encode functional components of T2S systems. Enzyme activity assays showed that the Xps system contributes to secretion of proteases and xylanases. We identified the virulence-associated xylanase XynC as a substrate of the Xps system. However, homologs of known T2S substrates from other Xanthomonas spp. are not secreted by the T2S systems from Xcv. Thus, T2S systems from Xanthomonas spp. appear to differ significantly in their substrate specificities. • Transcript analyses revealed that expression of xps genes in Xcv is activated by HrpG and HrpX, key regulators of the T3S system. By contrast, expression of xynC and extracellular protease and xylanase activities are repressed by HrpG and HrpX, suggesting that components and substrates of the Xps system are differentially regulated.


Dual roles for the variable domain in protein trafficking and hostspecific recognition of Heterodera glycines CLE effector proteins Jianying Wang, Chris Lee, Amy Replogle, Sneha Joshi, Dmitry Korkin, Richard Hussey, Thomas J. Baum, Eric L. Davis, Xiaohong Wang and Melissa G. Mitchum

Summary Author for correspondence: Melissa G. Mitchum Tel: +1 573 882 6152 Email: goellnerm@missouri.edu

• Soybean cyst nematodes (Heterodera glycines) produce secreted effector proteins that function as peptide mimics of plant CLAVATA3 ⁄ ESR (CLE)-like peptides probably involved in the developmental reprogramming of root cells to form specialized feeding cells called syncytia.

New Phytologist (2010) 187: 1003–1017 doi: 10.1111/j.1469-8137.2010.03300.x

• The site of action and mechanism of delivery of CLE effectors to host plant cells by the nematode, however, have not been established. In this study, immunologic, genetic and biochemical approaches were used to reveal the localization and site of action of H. glycines-secreted CLE proteins in planta.

KEYWORDS: CLAVATA3, CLE, effector, ligand, molecular mimicry, peptide, soybean cyst nematode, syncytium.

• We present evidence indicating that the nematode CLE propeptides are delivered to the cytoplasm of syncytial cells, but ultimately function in the apoplast, consistent with their proposed role as ligand mimics of plant CLE peptides. We determined that the nematode 12-amino-acid CLE motif peptide is not sufficient for biological activity in vivo, pointing to an important role for sequences upstream of the CLE motif in function. • Genetic and biochemical analysis confirmed the requirement of the variable domain in planta for host-specific recognition and revealed a novel role in trafficking cytoplasmically delivered CLEs to the apoplast in order to function as ligand mimics.


The Pseudomonas syringae effector protein HopZ1a suppresses effector-triggered immunity Alberto P. Macho, Carlos M. Guevara, Pablo Tornero, Javier Ruiz-Albert and Carmen R. Beuzón

Summary • The Pseudomonas syringae pv syringae type III effector HopZ1a is a member of the HopZ effector family of cysteine-proteases that triggers immunity in Arabidopsis. This immunity is dependent on HopZ1a cysteine-protease activity, and independent of known resistance genes. We have previously shown that HopZ1a-triggered immunity is partially additive to that triggered by AvrRpt2. These partially additive effects could be caused by at least two mechanisms: their New Phytologist (2010) 187: 1018–1033 signalling pathways share a common element(s), or one effector interferes with doi: 10.1111/j.1469-8137.2010.03381.x the response triggered by the other. Author for correspondence: Carmen R. Beuzo´n Tel: + 34 952 131959 Email: cbl@uma.es

KEYWORDS: avirulence, effector, effectortriggered immunity, ETI suppression, plant defence, virulence.

• Here, we investigate the molecular basis for the partially additive effect displayed by AvrRpt2- and HopZ1a-triggered immunities, by analysing competitive indices, hypersensitive response and symptom induction, PR-1 accumulation, expression of PR genes, and systemic acquired resistance (SAR) induction. • Partially additive effects between these defence responses require HopZ1a cysteine-protease activity, and also take place between HopZ1a and AvrRps4 or AvrRpm1-triggered responses. We establish that HopZ1a-triggered immunity is independent of salicylic acid (SA), EDS1, jasmonic acid (JA) and ethylene (ET)dependent pathways, and show that HopZ1a suppresses the induction of PR-1 and PR-5 associated with P. syringae pv tomato (Pto)-triggered effector-triggered immunity (ETI)-like defences, AvrRpt2-triggered immunity, and Pto or Pto (avrRpt2) activation of SAR, and that suppression requires HopZ1a cysteineprotease activity. • Our results indicate that HopZ1a triggers an unusual resistance independent of known pathways and suppresses SA and EDS1-dependent resistance.


Intracellular expression of a host-selective toxin, ToxA, in diverse plants phenocopies silencing of a ToxA-interacting protein, ToxABP1 Viola A. Manning, Ashley L. Chu, Steven R. Scofield and Lynda M. Ciuffetti

Summary • ToxA, a host-selective toxin of wheat, can be detected within ToxA-sensitive mesophyll cells, where it localizes to chloroplasts and induces necrosis. Interaction of ToxA with the chloroplast-localized protein ToxABP1 has been implicated in this process. Therefore, we hypothesized that silencing of ToxABP1 in wheat would lead to a necrotic phenotype. Also, because ToxABP1 is highly conserved in plants, internal expression of ToxA in plants that do not normally internalize ToxA should New Phytologist (2010) 187: 1034–1047 result in cell death. Author for correspondence: Lynda M. Ciuffetti Tel: +1 541 7372188 Email: ciuffetL@science.oregonstate.edu

doi: 10.1111/j.1469-8137.2010.03363.x

KEYWORDS: Agrobacterium, Barley stripe mosaic virus, host-selective toxin, Nicotiana benthamiana, Pyrenophora tritici-repentis, ToxA, viral-induced gene silencing, wheat.

• Reduction of ToxABP1 expression was achieved using Barley stripe mosaic virus (BSMV)-mediated, viral-induced gene silencing. The BSMV system was modified for use as an internal expression vector for ToxA in monocots. Agrobacteriummediated expression of ToxA in a dicot (tobacco-Nicotiana benthamiana) was also performed. • Viral-induced gene silencing of ToxABP1 partially recapitulates the phenotype of ToxA treatment and wheat plants with reduced ToxABP1 also have reduced sensitivity to ToxA. When ToxA is expressed in ToxA-insensitive wheat, barley (Hordeum vulgare) and tobacco, cell death ensues. • ToxA accumulation in any chloroplast-containing cell is likely to result in cell death. Our data indicate that the ToxA–ToxABP1 interaction alters ToxABP1 function. This interaction is a critical, although not exclusive, component of the


Promoter elements of rice susceptibility genes are bound and activated by specific TAL effectors from the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae Patrick Römer, Sabine Recht, Tina Strauß, Janett Elsaesser, Sebastian Schornack, Jens Boch, Shiping Wang and Thomas Lahaye

Summary Author for correspondence: Thomas Lahaye Tel: +49 (0)89 2180 7470 Email: lahaye@bio.lmu.de

New Phytologist (2010) 187: 1048–1057 doi: 10.1111/j.1469-8137.2010.03217.x

KEYWORDS: AvrBs3, AvrXa27, Bs3, PthXo1, UPA (up-regulated by AvrBs3), Xa13, Xanthomonas.

• Plant pathogenic bacteria of the genus Xanthomonas inject transcription activatorlike effector (TALe) proteins that bind to and activate host promoters, thereby promoting disease or inducing plant defense. TALes bind to corresponding UPT (up-regulated by TALe) promoter boxes via tandemly arranged 34 ⁄ 35-amino acid repeats. Recent studies uncovered the TALe code in which two amino acid residues of each repeat define specific pairing to UPT boxes. • Here we employed the TALe code to predict potential UPT boxes in TALeinduced host promoters and analyzed these via b-glucuronidase (GUS) reporter and electrophoretic mobility shift assays (EMSA). • We demonstrate that the Xa13, OsTFX1 and Os11N3 promoters from rice are induced directly by the Xanthomonas oryzae pv. oryzae TALes PthXo1, PthXo6 and AvrXa7, respectively. We identified and functionally validated a UPT box in the corresponding rice target promoter for each TALe and show that box mutations suppress TALe-mediated promoter activation. Finally, EMSA demonstrate that code-predicted UPT boxes interact specifically with corresponding TALes. • Our findings show that variations in the UPT boxes of different rice accessions correlate with susceptibility or resistance of these accessions to the bacterial blight pathogen.


Suppression of the AvrBs1-specific hypersensitive response by the YopJ effector homolog AvrBsT from Xanthomonas depends on a SNF1 -related kinase Robert Szczesny, Daniela Büttner, Lucia Escolar, Sebastian Schulze, Anja Seiferth and Ulla Bonas

Summary Author for correspondence: Ulla Bonas Tel: +49 345 5526290 Email: ulla.bonas@genetik.uni-halle.de

• Pathogenicity of the Gram-negative plant pathogen Xanthomonas campestris pv. vesicatoria (Xcv) depends on a type III secretion system that translocates a cocktail of > 25 type III effector proteins into the plant cell.

• In this study, we identified the effector AvrBsT as a suppressor of specific plant defense. AvrBsT belongs to the YopJ ⁄ AvrRxv protein family, members of which New Phytologist (2010) 187: 1058–1074 are predicted to act as proteases and ⁄ or acetyltransferases. doi: 10.1111/j.1469-8137.2010.03346.x

KEYWORDS: AvrRxv, bacterial spot disease, effectors, pepper, protease, transacetylase, YopJ.

• AvrBsT suppresses the hypersensitive response (HR) that is elicited by the effector protein AvrBs1 from Xcv in resistant pepper plants. HR suppression occurs inside the plant cell and depends on a conserved predicted catalytic residue of AvrBsT. Yeast two-hybrid based analyses identified plant interaction partners of AvrBs1 and AvrBsT, including a putative regulator of sugar metabolism, SNF1related kinase 1 (SnRK1), as interactor of AvrBsT. Intriguingly, gene silencing experiments revealed that SnRK1 is required for the induction of the AvrBs1specific HR. • We therefore speculate that SnRK1 is involved in the AvrBsT-mediated suppression of the AvrBs1-specific HR.


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Effectors in plant–microbe interactions