Virtual Issue Articles: Effectors of biotrophic fungi and oomycetes. pathogenicity factors and triggers of host resistance Dodds, PN, Rafiqi, M, Gan, PHP, Hardham, AR, Jones, DA, Ellis, JG. Effector wisdom Lee, AH-Y, Petre, B, Joly, DL. Evolution of the type III secretion system and its effectors in plant-microbe interactions McCann, HC, Guttman, DS. The Pseudomonas syringae type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9 Block, A, Toruño, TY, Elowsky, CG, Zhang, C, Steinbrenner, J, Beynon, J, Alfano, JR. The bacterial effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal immunity Lozano-Durán, R, Bourdais, G, He, SY, Robatzek, S. The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity Hann, DR, Domínguez-Ferreras, A, Motyka, V, Dobrev, PI, Schornack, S, Jehle, A, Felix, G, Chinchilla, D, Rathjen, JP, Boller, T. HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens Sohn, KH, Saucet, SB, Clarke, CR, Vinatzer, BA, O'Brien, HE, Guttman, DS, Jones, JDG.

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity Schulze, S, Kay, S, Büttner, D, Egler, M, Eschen-Lippold, L, Hause, G, Krüger, A, Lee, J, Müller, O, Scheel, D, Szczesny, R, Thieme, F, Bonas, U. SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, requires resistance-gene-mediated signalling to activate cell death in the model plant Nicotiana benthamiana Üstün, S, Müller, P, Palmisano, R, Hensel, M, Börnke, F. Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae Verdier, V, Triplett, LR, Hummel, AW, Corral, R, Cernadas, RA, Schmidt, CL, Bogdanove, AJ, Leach, JE. Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae Streubel, J, Pesce, C, Hutin, M, Koebnik, R, Boch, J, Szurek, B. Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak Hummel, AW, Doyle, EL, Bogdanove, AJ. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease de Lange, O, Schreiber, T, Schandry, N, Radeck, J, Braun, KH, Koszinowski, J, Heuer, H, Strauß, A, Lahaye, T.

Virtual Issue Articles:

Oomycete and fungal effector entry, a microbial Trojan horse Kale, SD. The Phytophthora parasitica RXLR effector Penetration-Specific Effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology Evangelisti, E, Govetto, B, Minet-Kebdani, N, Kuhn, M-L, Attard, A, Ponchet, M, Panabières, F, Gourgues, M. The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death Yu, X, Tang, J, Wang, Q, Ye, W, Tao, K, Duan, S, Lu, C, Yang, X, Dong, S, Zheng, X, Wang, Y. Towards population genomics of effector–effector target interactions Terauchi, R, Yoshida, K. The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa. Balesdent, M-H, Fudal, I, Ollivier, B, Bally, P, Grandaubert, J, Eber, F, Chèvre, A-M, Leflon, M, Rouxel, T. The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1. Larkan, NJ, Lydiate, DJ, Parkin, IAP, Nelson, MN, Epp, DJ, Cowling, WA, Rimmer, SR, Borhan, MH. Global diversity and distribution of three necrotrophic effectors in Phaeosphaeria nodorum and related species McDonald, MC, Oliver, RP, Friesen, TL, Brunner, PC, McDonald, BA.

The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat. Du Fall, LA, Solomon, PS. Dual roles for the variable domain in protein trafficking and host-specific recognition of Heterodera glycines CLE effector proteins Wang, J, Lee, C, Replogle, A, Joshi, S, Korkin, D, Hussey, R, Baum, TJ, Davis, EL, Wang, X, Mitchum, MG. Nematode effector proteins: an emerging paradigm of parasitism Mitchum, MG, Hussey, RS, Baum, TJ, Wang, X, Elling, AA, Wubben, M, Davis, EL. Cover Image: Micrograph visualizing Bimolecular fluorescence complementation (BiFC) analysis of the interaction between the bacterial (Pseudomonas syringae) effector HopD1 and its plant target, the Arabidopsis thaliana transcription factor NTL9. Yellow fluorescence signifies the complemented YFP molecule at the plant endoplasmic reticulum, the red signal is the result of chlorophyll autofluorescence. Picture taken from Block et al. (2013).

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Introduction Pathogen effectors are small secreted proteins which can be found in the apoplastic space or in the cytoplasm of plant cells as a result of infection by parasites. Effectors can be involved in reprogramming host cells by either suppressing plant defence responses and/or diverting nutrient flow towards the microbial intruder. The growth in literature devoted to effectors is clear in the collection of papers presented here. The New Phytologist Trust has particularly supported research in this area through the organisation of two symposia with an emphasis on effectors in plant–microbe interactions: the 22nd New Phytologist Symposium in 2009 and the 30th New Phytologist Symposium in 2012.

Scheme representing the logo of the 22nd New Phytologist Symposium on effectors in plant–microbe interactions (held in Versailles, France, September 2009), symbolizing effector-mediated plant attack by fungi/oomycetes (upper left), bacteria (upper right), viruses (lower left) and nematodes (lower right).

Review

Effectors of biotrophic fungi and oomycetes: pathogenicity factors and triggers of host resistance

Dodds, PN, Rafiqi, M, Gan, PHP, Hardham, AR, Jones, DA, Ellis, JG.

Summary

New Phytologist (2009) 183: 993–999 doi: 10.1111/j.1469-8137.2009.02922.x Key words: avirulence, biotroph, effector proteins, haustoria, oomycete, resistance, rust

Many biotrophic fungal and oomycete pathogens share a common infection process involving the formation of haustoria, which penetrate host cell walls and form a close association with plant membranes. Recent studies have identified a class of pathogenicity effector proteins from these pathogens that is transferred into host cells from haustoria during infection. This insight stemmed from the identification of avirulence (Avr) proteins from these pathogens that are recognized by intracellular host resistance (R) proteins. Oomycete effectors contain a conserved translocation motif that directs their uptake into host cells independently of the pathogen, and is shared with the human malaria pathogen. Genome sequence information indicates that oomycetes may express several hundred such host-translocated effectors. Elucidating the transport mechanism of fungal and oomycete effectors and their roles in disease offers new opportunities to understand how these pathogens are able to manipulate host cells to establish a parasitic relationship and to develop new disease-control measures.

Effector wisdom

Lee, AH-Y, Petre, B, Joly, DL.

New Phytologist (2013) 197: 375–377 doi: 10.1111/nph.12058 Key words: effectors, genomics, immunity, pathogens, plant-associated organisms, plant–microbe interactions, secretion, targets

Many organisms such as bacteria, fungi, oomycetes, nematodes and insects grow, feed and/or reproduce in close association with plant hosts. To establish such intimate interactions, symbionts (either mutualistic or parasitic) secrete effectors into host tissues, which are molecules that modulate plant cell structures and processes (Win et al., 2012a). This last decade, advances in genomics have revealed that symbionts possess dozens to hundreds of effectors. Currently, the field is moving rapidly from effector identification towards effector characterization, which provides a better understanding of how these effectors promote the establishment of a successful relationship with host plants. The 30th New Phytologist Symposium clearly illustrated this theme, as an international panel of c. 150 scientists was brought together to discuss current efforts to decipher effector functions within a wide range of biological systems.

Meeting Report

McCann, HC, Guttman, DS.

Evolution of the type III secretion system and its effectors in plant–microbe interactions

Summary

New Phytologist (2008) 177: 33–47 doi: 10.1111/j.1469-8137.2007.02293.x Key words: bacterial pathogens, evolution, plant–pathogen interactions, Pseudomonas syringae, type III secreted effector protein (T3SE), type III secretion system (T3SS), Xanthomonas

Many bacterial plant pathogens require the type III secretion system (T3SS) and its effector proteins (T3SEs) to invade and extract nutrients from their hosts successfully. While the molecular function of this system is being studied intensively, we know comparatively little about the evolutionary and ecological pressures governing its fate over time, and even less about the detailed mechanisms underlying and driving complex T3SS-mediated coevolutionary dynamics. In this review we summarize our current understanding of how host–pathogen interactions evolve, with a particular focus on the T3SS of bacterial plant pathogens. We explore the evolutionary origins of the T3SS relative to the closely related flagellar system, and investigate the evolutionary pressures on this secretion and translocation apparatus. We examine the evolutionary forces acting on T3SEs, and compare the support for vertical descent with modification of these virulence-associated systems (pathoadaptation) vs horizontal gene transfer. We address the evolutionary origins of T3SEs from the perspective of both the evolutionary mechanisms that generate new effectors, and the mobile elements that may be the source of novel genetic material. Finally, we propose a number of questions raised by these studies, which may serve to guide our thinking about these complex processes.

Review

Research

The Pseudomonas syringae type III effector HopD1 suppresses effector-triggered immunity, localizes to the endoplasmic reticulum, and targets the Arabidopsis transcription factor NTL9

Block, A, Toruño, TY, Elowsky, CG, Zhang, C, Steinbrenner, J, Beynon, J, Alfano, JR.

Summary

New Phytologist (2014) 201: 1358–1370 doi: 10.1111/nph.12626 Key words: bacterial pathogens, plant defense, plant disease, plant immunity, type III effector

• Pseudomonas syringae type III effectors are known to suppress plant immunity to promote bacterial virulence. However, the activities and targets of these effectors are not well understood.

• We used genetic, molecular, and cell biology methods to characterize the activities, localization, and target of the HopD1 type III effector in Arabidopsis.

• HopD1 contributes to P. syringae virulence in Arabidopsis and reduces effector-triggered immunity (ETI) responses but not pathogen-associated molecular pattern-triggered immunity (PTI) responses. Plants expressing HopD1 supported increased growth of ETI-inducing P. syringae strains compared with wild-type Arabidopsis. We show that HopD1 interacts with the membrane-tethered Arabidopsis transcription factor NTL9 and demonstrate that this interaction occurs at the endoplasmic reticulum (ER). A P. syringae hopD1 mutant and ETI-inducing P. syringae strains exhibited enhanced growth on Arabidopsis ntl9 mutant plants. Conversely, growth of P. syringae strains was reduced in plants expressing a constitutively active NTL9 derivative, indicating that NTL9 is a positive regulator of plant immunity. Furthermore, HopD1 inhibited the induction of NTL9-regulated genes during ETI but not PTI.

• HopD1 contributes to P. syringae virulence in part by targeting NTL9, resulting in the suppression of ETI responses but not PTI responses and the promotion of plant pathogenicity.

The bacterial effector HopM1 suppresses PAMP-triggered oxidative burst and stomatal immunity

Lozano-Durán, R, Bourdais, G, He, SY, Robatzek, S.

Summary

New Phytologist (2014) 202: 259 - 269 doi: 10.1111/nph.12651 Key words: 14-3-3, effector, HopM1, immunity, oxidative burst, PAMP/MAMP, stomata, TFT1

• Successful pathogens counter immunity at multiple levels, mostly through the action of effectors. Pseudomonas syringae secretes c. 30 effectors, some of which have been shown to inhibit plant immunity triggered upon perception of conserved pathogen-associated molecular patterns (PAMPs). One of these is HopM1, which impairs late immune responses through targeting the vesicle trafficking-related AtMIN7 for degradation.

• Here, we report that in planta expressed HopM1 suppresses two early PAMP-triggered responses, the oxidative burst and stomatal immunity, both of which seem to require proteasomal function but are independent of AtMIN7. Notably, a 14-3-3 protein, GRF8/AtMIN10, was found previously to be a target of HopM1 in vivo, and expression of HopM1 mimics the effect of chemically and genetically disrupting 14-3-3 function.

• Our data further show that the function of 14-3-3 proteins is required for PAMP-triggered oxidative burst and stomatal immunity, and chemical-mediated disruption of the 14-3-3 interactions with their client proteins restores virulence of a HopM1-deficient P. syringae mutant, providing a link between HopM1 and the involvement of 14-3-3 proteins in plant immunity.

• Taken together, these results unveil the impact of HopM1 on the PAMP-triggered oxidative burst and stomatal immunity in an AtMIN7-independent manner, most likely acting at the function of (a) 14-3-3 protein(s).

Research

The Pseudomonas type III effector HopQ1 activates cytokinin signaling and interferes with plant innate immunity

Hann, DR, Domínguez-Ferreras, A, Motyka, V, Dobrev, PI, Schornack, S, Jehle, A, Felix, G, Chinchilla, D, Rathjen, JP, Boller, T.

Summary

New Phytologist (2014) 201: 585 - 598 doi: 10.1111/nph.12544 Key words: cytokinin, flagellin, FLS2, HopQ1, innate immunity, type III effector

• We characterized the molecular function of the Pseudomonas syringae pv. tomato DC3000 (Pto) effector HopQ1.

• In silico studies suggest that HopQ1 might possess nucleoside hydrolase activity based on the presence of a characteristic aspartate motif. Transgenic Arabidopsis lines expressing HopQ1 or HopQ1 aspartate mutant variants were characterized with respect to flagellin triggered immunity, phenotype and changes in phytohormone content by high-performance liquid chromatography-MS (HPLC-MS).

• We found that HopQ1, but not its aspartate mutants, suppressed all tested immunity marker assays. Suppression of immunity was the result of a lack of the flagellin receptor FLS2, whose gene expression was abolished by HopQ1 in a promoter-dependent manner. Furthermore, HopQ1 induced cytokinin signaling in Arabidopsis and the elevation in cytokinin signaling appears to be responsible for the attenuation of FLS2 expression.

• We conclude that HopQ1 can activate cytokinin signaling and that moderate activation of cytokinin signaling leads to suppression of FLS2 accumulation and thus defense signaling.

Research

Research

HopAS1 recognition significantly contributes to Arabidopsis nonhost resistance to Pseudomonas syringae pathogens

Sohn, KH, Saucet, SB, Clarke, CR, Vinatzer, BA, O'Brien, HE, Guttman, DS, Jones, JDG.

Summary

New Phytologist (2012) 193: 58 - 66 doi: 10.1111/j.1469-8137.2011.03950.x Key words: Arabidopsis, effector-triggered immunity, nonhost resistance, Pseudomonas, type III effector

• Plant immunity is activated by sensing either conserved microbial signatures, called pathogen/microbe-associated molecular patterns (P/MAMPs), or specific effectors secreted by pathogens. However, it is not known why most microbes are nonpathogenic in most plant species.

• Nonhost resistance (NHR) consists of multiple layers of innate immunity and protects plants from the vast majority of potentially pathogenic microbes. Effector-triggered immunity (ETI) has been implicated in race-specific disease resistance. However, the role of ETI in NHR is unclear.

• Pseudomonas syringae pv. tomato (Pto) T1 is pathogenic in tomato (Solanum lycopersicum) yet nonpathogenic in Arabidopsis. Here, we show that, in addition to the type III secretion system (T3SS)-dependent effector (T3SE) avrRpt2, a second T3SE of Pto T1, hopAS1, triggers ETI in nonhost Arabidopsis.

• hopAS1 is broadly present in P. syringae strains, contributes to virulence in tomato, and is quantitatively required for Arabidopsis NHR to Pto T1. Strikingly, all tested P. syringae strains that are pathogenic in Arabidopsis carry truncated hopAS1 variants of forms, demonstrating that HopAS1-triggered immunity plays an important role in Arabidopsis NHR to a broad-range of P. syringae strains.

Analysis of new type III effectors from Xanthomonas uncovers XopB and XopS as suppressors of plant immunity

Schulze, S, Kay, S, Büttner, D, Egler, M, Eschen-Lippold, L, Hause, G, Krüger, A, Lee, J, Müller, O, Scheel, D, Szczesny, R, Thieme, F, Bonas, U.

Summary

New Phytologist (2012) 195: 894 - 911 doi: 10.1111/j.1469-8137.2012.04210.x Key words: viral spot disease, Capsicum annuum (pepper), cell death suppression, effector, HpaB, type III secretion, vesicle trafficking, Xanthomonas campestris

• The pathogenicity of the Gram-negative plant-pathogenic bacterium Xanthomonas campestris pv. vesicatoria (Xcv) is dependent on type III effectors (T3Es) that are injected into plant cells by a type III secretion system and interfere with cellular processes to the benefit of the pathogen.

• In this study, we analyzed eight T3Es from Xcv strain 85-10, six of which were newly identified effectors. Genetic studies and protoplast expression assays revealed that XopB and XopS contribute to disease symptoms and bacterial growth, and suppress pathogen-associated molecular pattern (PAMP)-triggered plant defense gene expression.

• In addition, XopB inhibits cell death reactions induced by different T3Es, thus suppressing defense responses related to both PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI).

• XopB localizes to the Golgi apparatus and cytoplasm of the plant cell and interferes with eukaryotic vesicle trafficking. Interestingly, a XopB point mutant derivative was defective in the suppression of ETI-related responses, but still interfered with vesicle trafficking and was only slightly affected with regard to the suppression of defense gene induction. This suggests that XopB-mediated suppression of PTI and ETI is dependent on different mechanisms that can be functionally separated.

Research

SseF, a type III effector protein from the mammalian pathogen Salmonella enterica, requires resistance-gene-mediated signalling to activate cell death in the model plant Nicotiana benthamiana

Üstün, S, Müller, P, Palmisano, R, Hensel, M, Börnke, F.

Summary

New Phytologist (2012) 194: 1046 - 1060 doi: 10.1111/j.1469-8137.2012.04124.x Key words: hypersensitive response (HR), plant–bacteria interaction, R protein, Salmonella, SseF, type III effector protein (T3E)

• Type III effector proteins (T3Es) of many Gram-negative pathogenic bacteria manipulate highly conserved cellular processes, indicating conservation in virulence mechanisms during the infection of hosts of divergent evolutionary origin.

• In order to identify conserved effector functions, we used a cross-kingdom approach in which we expressed selected T3Es from the mammalian pathogen Salmonella enterica in leaves of Nicotiana benthamiana and searched for possible virulence or avirulence phenotypes.

• We show that the T3E SseF of S. enterica triggers hypersensitive response (HR)-like symptoms, a hallmark of effector-triggered immunity in plants, either when transiently expressed in leaves of N. benthamiana by Agrobacterium tumefaciens infiltration or when delivered by Xanthomonas campestris pv vesicatoria (Xcv) through the type III secretion system. The ability of SseF to elicit HR-like symptoms was lost upon silencing of suppressor of G2 allele of skp1 (SGT1), indicating that the S. enterica T3E is probably recognized by an R protein in N. benthamiana. Xcv translocating an AvrRpt2–SseF fusion protein was restricted in multiplication within leaves of N. benthamiana. Bacterial growth was not impaired but symptom development was rather accelerated in a compatible interaction with susceptible pepper (Capsicum annuum) plants.

• We conclude that the S. enterica T3E SseF is probably recognized by the plant immune system in N. benthamiana, resulting in effector-triggered immunity.

Research

Transcription activator-like (TAL) effectors targeting OsSWEET genes enhance virulence on diverse rice (Oryza sativa) varieties when expressed individually in a TAL effector-deficient strain of Xanthomonas oryzae

Verdier, V, Triplett, LR, Hummel, AW, Corral, R, Cernadas, RA, Schmidt, CL, Bogdanove, AJ, Leach, JE.

Summary

New Phytologist (2012) 196: 1197 - 1207 doi: 10.1111/j.1469-8137.2012.04367.x Key words: OryzaSNP, OsSWEET, transcription activator-like (TAL) effectors, Xanthomonas oryzae

• Genomes of the rice (Oryza sativa) xylem and mesophyll pathogens Xanthomonas oryzae pv. oryzae (Xoo) and pv. oryzicola (Xoc) encode numerous secreted transcription factors called transcription activator-like (TAL) effectors. In a few studied rice varieties, some of these contribute to virulence by activating corresponding host susceptibility genes. Some activate disease resistance genes. The roles of X. oryzae TAL effectors in diverse rice backgrounds, however, are poorly understood.

• Xoo TAL effectors that promote infection by activating SWEET sucrose transporter genes were expressed in TAL effector-deficient X. oryzae strain X11-5A, and assessed in 21 rice varieties. Some were also tested in Xoc on variety Nipponbare. Several Xoc TAL effectors were tested in X11-5A on four rice varieties.

• Xoo TAL effectors enhanced X11-5A virulence on most varieties, but to varying extents depending on the effector and variety. SWEET genes were activated in all tested varieties, but increased virulence did not correlate with activation level. SWEET activators also enhanced Xoc virulence on Nipponbare. Xoc TAL effectors did not alter X11-5A virulence.

• SWEET-targeting TAL effectors contribute broadly and non-tissue-specifically to virulence in rice, and their function is affected by host differences besides target sequences. Further, the utility of X11-5A for characterizing individual TAL effectors in rice was established.

Research

Five phylogenetically close rice SWEET genes confer TAL effector-mediated susceptibility to Xanthomonas oryzae pv. oryzae

Streubel, J, Pesce, C, Hutin, M, Koebnik, R, Boch, J, Szurek, B.

Summary

New Phytologist (2013) 200: 808 - 819 doi: 10.1111/nph.12411 Key words: designer TALE, plant pathogen, plant resistance, rice, TALE, type-III effector, virulence, OsSWEET

• Bacterial plant-pathogenic Xanthomonas strains translocate transcription activator-like (TAL) effectors into plant cells to function as specific transcription factors. Only a few plant target genes of TAL effectors have been identified, so far. Three plant SWEET genes encoding putative sugar transporters are known to be induced by TAL effectors from rice-pathogenic Xanthomonas oryzae pv. oryzae (Xoo).

• We predict and validate that expression of OsSWEET14 is induced by a novel TAL effector, Tal5, from an African Xoo strain. Artificial TAL effectors (ArtTALs) were constructed to individually target 20 SWEET orthologs in rice. They were used as designer virulence factors to study which rice SWEET genes can support Xoo virulence.

• The Tal5 target box differs from those of the already known TAL effectors TalC, AvrXa7 and PthXo3, which also induce expression of OsSWEET14, suggesting evolutionary convergence on key targets. ArtTALs efficiently complemented an Xoo talC mutant, demonstrating that specific induction of OsSWEET14 is the key target of TalC. ArtTALs that specifically target individual members of the rice SWEET family revealed three known and two novel SWEET genes to support bacterial virulence.

• Our results demonstrate that five phylogenetically close SWEET proteins, which presumably act as sucrose transporters, can support Xoo virulence.

Research

Addition of transcription activator-like effector binding sites to a pathogen strain-specific rice bacterial blight resistance gene makes it effective against additional strains and against bacterial leaf streak

Hummel, AW, Doyle, EL, Bogdanove, AJ.

Summary

New Phytologist (2012) 195: 883 - 893 doi: 10.1111/j.1469-8137.2012.04216.x Key words: effector-triggered immunity, gene regulation, plant disease resistance, R gene, resistance gene, TAL effector, Xanthomonas

• Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it.

• We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99A and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc).

• Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99A, a PXO99A derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences.

• Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

Research

Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease

de Lange, O, Schreiber, T, Schandry, N, Radeck, J, Braun, KH, Koszinowski, J, Heuer, H, Strauß, A, Lahaye, T.

Summary

New Phytologist (2013) 199: 773 - 786 doi: 10.1111/nph.12324 Key words: AvrBs3 (avirulence protein triggering Bs3 resistance), Brg11(hrpB-regulated 11), designer transcription activator-like effector (TALE), GMI1000, Ralstonia solanacearum, Ralstonia transcription activator-like effector (TALE)-like (RTL), repeat variable diresidue (RVD), transcription activator-like (TAL) effectors

• Ralstonia solanacearum is a devastating bacterial phytopathogen with a broad host range. Ralstonia solanacearum injected effector proteins (Rips) are key to the successful invasion of host plants. We have characterized Brg11(hrpB-regulated 11), the first identified member of a class of Rips with high sequence similarity to the transcription activator-like (TAL) effectors of Xanthomonas spp., collectively termed RipTALs.

• Fluorescence microscopy of in planta expressed RipTALs showed nuclear localization. Domain swaps between Brg11 and Xanthomonas TAL effector (TALE) AvrBs3 (avirulence protein triggering Bs3 resistance) showed the functional interchangeability of DNA-binding and transcriptional activation domains. PCR was used to determine the sequence of brg11 homologs from strains infecting phylogenetically diverse host plants.

• Brg11 localizes to the nucleus and activates promoters containing a matching effector-binding element (EBE). Brg11 and homologs preferentially activate promoters containing EBEs with a 5′ terminal guanine, contrasting with the TALE preference for a 5′ thymine.

• Brg11 and other RipTALs probably promote disease through the transcriptional activation of host genes. Brg11 and the majority of homologs identified in this study were shown to activate similar or identical target sequences, in contrast to TALEs, which generally show highly diverse target preferences. This information provides new options for the engineering of plants resistant to R. solanacearum.

Research

Oomycete and fungal effector entry, a microbial Trojan horse

Kale, SD.

Summary

New Phytologist (2012) 193: 885 - 881 doi: 10.1111/j.1469-8137.2011.03968.x Key words: effectors, endocytosis, fungi, mutualists, oomycetes, pathogens, phospholipids, RXLR and RXLR-like

Oomycete and fungal symbionts have significant impacts on most commercially important crop and forest species, and on natural ecosystems, both negatively as pathogens and positively as mutualists. Symbiosis may be facilitated through the secretion of effector proteins, some of which modulate a variety of host defense mechanisms. A subset of these secreted proteins are able to translocate into host cells. In the oomycete pathogens, two conserved N-terminal motifs, RXLR and dEER, mediate translocation of effector proteins into host cells independent of any pathogen-encoded machinery. An expanded ‘RXLR-like’ motif [R/K/H]X[L/M/I/F/Y/W]X has been used to identify functional translocation motifs in host-cell-entering fungal effector proteins from pathogens and a mutualist. The RXLR-like translocation motifs were required for the fungal effectors to enter host cells in the absence of any pathogen-encoded machinery. Oomycete and fungal effectors with RXLR and RXLR-like motifs can bind phospholipids, specifically phosphatidylinositol-3-phosphate (PtdIns-3-P). Effector-PtdIns-3-P binding appears to mediate cell entry via lipid raft-mediated endocytosis, and could be blocked by sequestering cell surface PtdIns-3-P or by utilizing inositides that competitively inhibit effector binding to PtdIns-3-P. These findings suggest that effector blocking technologies could be developed and utilized in a variety of important crop species against a broad spectrum of plant pathogens.

Review

Research

The Phytophthora parasitica RXLR effector Penetration-Specific Effector 1 favours Arabidopsis thaliana infection by interfering with auxin physiology

Evangelisti, E, Govetto, B, Minet-Kebdani, N, Kuhn, M-L, Attard, A, Ponchet, M, Panabières, F, Gourgues, M.

Summary

New Phytologist (2013) 199: 476 - 489 doi: 10.1111/nph.12270 Key words: Arabidopsis thaliana, auxin, effector, oomycete, Phytophthora parasitica

• Pathogenic oomycetes have evolved RXLR effectors to thwart plant defense mechanisms and invade host tissues. We analysed the function of one of these effectors (Penetration-Specific Effector 1 (PSE1)) whose transcript is transiently accumulated during penetration of host roots by the oomycete Phytophthora parasitica.

• Expression of PSE1 protein in tobacco (Nicotiana tabacum and Nicotiana benthamiana) leaves and in Arabidopsis thaliana plants was used to assess the role of this effector in plant physiology and in interactions with pathogens. A pharmacological approach and marker lines were used to charcterize the A. thaliana phenotypes.

• Expression of PSE1 in A. thaliana led to developmental perturbations associated with low concentrations of auxin at the root apex. This modification of auxin content was associated with an altered distribution of the PIN4 and PIN7 auxin efflux carriers. The PSE1 protein facilitated plant infection: it suppressed plant cell death activated by Pseudomonas syringae avirulence gene AvrPto and Phytophthora cryptogea elicitin cryptogein in tobacco and exacerbated disease symptoms upon inoculation of transgenic A. thaliana plantlets with P. parasitica in an auxin-dependant manner.

• We propose that P. parasitica secretes the PSE1 protein during the penetration process to favour the infection by locally modulating the auxin content. These results support the hypothesis that effectors from plant pathogens may act on a limited set of targets, including hormones.

Research

The RxLR effector Avh241 from Phytophthora sojae requires plasma membrane localization to induce plant cell death

Yu, X, Tang, J, Wang, Q, Ye, W, Tao, K, Duan, S, Lu, C, Yang, X, Dong, S, Zheng, X, Wang, Y.

Summary

New Phytologist (2012) 196: 247 - 260 doi: 10.1111/j.1469-8137.2012.04241.x Key words: cell death, localization, Phytophthora sojae, plasma membrane, RxLR effector, virulence

• The Phytophthora sojae genome encodes hundreds of RxLR effectors predicted to manipulate various plant defense responses, but the molecular mechanisms involved are largely unknown. Here we have characterized in detail the P. sojae RxLR effector Avh241.

• To determine the function and localization of Avh241, we transiently expressed it on different plants. Silencing of Avh241 in P. sojae, we determined its virulence during infection. Through the assay of promoting infection by Phytophthora capsici to Nicotiana benthamiana, we further confirmed this virulence role.

• Avh241 induced cell death in several different plants and localized to the plant plasma membrane. An N-terminal motif within Avh241 was important for membrane localization and cell death-inducing activity. Two mitogen-activated protein kinases, NbMEK2 and NbWIPK, were required for the cell death triggered by Avh241 in N. benthamiana. Avh241 was important for the pathogen’s full virulence on soybean. Avh241 could also promote infection by P. capsici and the membrane localization motif was not required to promote infection.

• This work suggests that Avh241 interacts with the plant immune system via at least two different mechanisms, one recognized by plants dependent on subcellular localization and one promoting infection independent on membrane localization.

Towards population genomics of effector-effector target interactions

Terauchi, R, Yoshida, K.

Summary

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

Review

Research

The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa

Balesdent, M-L, Fudal, I, Ollivier, B, Bally, P, Grandaubert, J, Eber, F, Chèvre, A-M, Leflon, M, Rouxel, T.

Summary

New Phytologist (2013) 198: 887 - 898 doi: 10.1111/nph.12178 Key words: avirulence gene, Brassica napus, Brassica rapa, dispensable or B chromosomes, effector, fitness, Leptosphaeria maculans

• Phytopathogenic fungi frequently contain dispensable chromosomes, some of which contribute to host range or pathogenicity. In Leptosphaeria maculans, the stem canker agent of oilseed rape (Brassica napus), the minichromosome was previously suggested to be dispensable, without evidence for any role in pathogenicity.

• Using genetic and genomic approaches, we investigated the inheritance and molecular determinant of an L. maculans–Brassica rapa incompatible interaction.

• Single gene control of the resistance was found, while all markers located on the L. maculans minichromosome, absent in the virulent parental isolate, co-segregated with the avirulent phenotype. Only one candidate avirulence gene was identified on the minichromosome, validated by complementation experiments and termed AvrLm11. The minichromosome was frequently lost following meiosis, but the frequency of isolates lacking it remained stable in field populations sampled at a 10-yr time interval, despite a yearly sexual stage in the L. maculans life cycle.

• This work led to the cloning of a new ‘lost in the middle of nowhere’ avirulence gene of L. maculans, interacting with a B. rapa resistance gene termed Rlm11 and introgressed into B. napus. It demonstrated the dispensability of the L. maculans minichromosome and suggested that its loss generates a fitness deficit.

Research

The Brassica napus blackleg resistance gene LepR3 encodes a receptor-like protein triggered by the Leptosphaeria maculans effector AVRLM1

Larkan, NJ, Lydiate, DJ, Parkin, IAP, Nelson, MN, Epp, DJ, Cowling, WA, Rimmer, SR, Borhan, MH.

Summary

New Phytologist (2013) 197: 595 - 605 doi: 10.1111/nph.12043 Key words: Arabidopsis thaliana, blackleg resistance, Brassica napus (canola/oilseed rape), Brassica rapa, collinearity, Leptosphaeria maculans, receptor-like protein

• LepR3, found in the Brassica napus cv ‘Surpass 400’, provides race-specific resistance to the fungal pathogen Leptosphaeria maculans, which was overcome after great devastation in Australia in 2004. We investigated the LepR3 locus to identify the genetic basis of this resistance interaction.

• We employed a map-based cloning strategy, exploiting collinearity with the Arabidopsis thaliana and Brassica rapa genomes to enrich the map and locate a candidate gene. We also investigated the interaction of LepR3 with the L. maculans avirulence gene AvrLm1 using transgenics.

• LepR3 was found to encode a receptor-like protein (RLP). We also demonstrated that avirulence towards LepR3 is conferred by AvrLm1, which is responsible for both the Rlm1 and LepR3-dependent resistance responses in B. napus.

• LepR3 is the first functional B. napus disease resistance gene to be cloned. AvrLm1's interaction with two independent resistance loci, Rlm1 and LepR3, highlights the need to consider redundant phenotypes in ‘gene-for-gene’ interactions and offers an explanation as to why LepR3 was overcome so rapidly in parts of Australia.

Research

Global diversity and distribution of three necrotrophic effectors in Phaeosphaeria nodorum and related species

McDonald, MC, Oliver, RP, Friesen, TL, Brunner, PC, McDonald, BA.

Summary

New Phytologist (2013) 199, 241 - 251 doi: 10.1111/nph.12257 Key words: co-evolution, fungal effectors, NB-LRR, necrotrophic effectors, Phaeosphaeria nodorum, plant pathogen, population genetics

• Population genetic and phylogenetic studies have shown that Phaeosphaeria nodorum is a member of a species complex that probably shares its center of origin with wheat (Triticum aestivum and Triticum durum). We examined the evolutionary histories of three known necrotrophic effectors (NEs) produced by P. nodorum and compared them with neutral loci.

• We screened over 1000 individuals for the presence/absence of each effector and assigned each individual to a multi-effector genotype. Diversity at each NE locus was assessed by sequencing c. 200 individuals for each locus.

• We found significant differences in effector frequency among populations. We propose that these differences reflect the presence/absence of the corresponding susceptibility gene in wheat cultivars. The population harboring the highest sequence diversity was different for each effector locus and never coincided with populations harboring the highest diversity at neutral loci. Coalescent and phylogenetic analyses showed a discontinuous presence of all three NEs among nine closely related Phaeosphaeria species. Only two of the nine species were found to harbor NEs.

• We present evidence that the three described NEs of P. nodorum were transmitted to its sister species, Phaeosphaeria avenaria tritici 1, via interspecific hybridization.

Research

The necrotrophic effector SnToxA induces the synthesis of a novel phytoalexin in wheat

Du Fall, LA, Solomon, PS.

Summary

New Phytologist (2013) 200: 185 - 200 doi: 10.1111/nph.12356 Key words: effector, plant defence secondary metabolism, serotonin, SnToxA, Stagonospora nodorum, wheat (Triticum aestivum)

• Stagonospora nodorum and Pyrenophora tritici-repentis produce the effector ToxA that interacts with the dominant susceptibility gene in wheat, Tsn1. However, the way in which ToxA induces cell death and causes disease is unclear. Here, we performed comprehensive metabolite profiling of ToxA-infiltrated wheat (Triticum aestivum) to observe the secondary metabolite response to this effector.

• A strong induction of secondary metabolism subsequent to SnToxA infiltration was observed, including the monoamine serotonin. We established a novel role for serotonin as a phytoalexin in wheat and demonstrated that serotonin strongly inhibited sporulation of S. nodorum. Microscopy revealed that serotonin interferes with spore formation and maturation within pycnidial structures of the fungus. Subsequent analysis of S. nodorum exposed to serotonin revealed metabolites changes previously associated with sporulation, including trehalose and alternariol. Furthermore, we identified significantly lower concentrations of serotonin during infection compared with infiltration with ToxA, providing evidence that S. nodorum may suppress plant defence.

• This is the first study demonstrating induction of plant secondary metabolites in response to a necrotrophic effector that have significant antifungal potential against the pathogen. While it is generally accepted that necrotrophs exploit host cell responses, the current research strengthens the notion that necrotrophs require mechanisms to overcome plant defence to survive initial stages of infection.

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

Wang, J, Lee, C, Replogle, A, Joshi, S, Korkin, D, Hussey, R, Baum, TJ, Davis, EL, Wang, X, Mitchum, MG.

Summary

New Phytologist (2010) 200: 222 - 228 doi: 10.1111/j.1469-8137.2010.03300.x Key words: CLAVATA3, CLE, effector, ligand, molecular mimicry, peptide, soybean cyst nematode, syncytium

• 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.

• 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.

• 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.

Research

Nematode effector proteins: an emerging paradigm of parasitism

Mitchum, MG, Hussey, RS, Baum, TJ, Wang, X, Elling, AA, Wubben, M, Davis, EL.

Summary

New Phytologist (2013) 199: 879 - 894 doi: 10.1111/nph.12323 Key words: cyst, effector, giant cell, nematode, parasitism, reniform, root-knot, syncytium

Phytonematodes use a stylet and secreted effectors to modify host cells and ingest nutrients to support their growth and development. The molecular function of nematode effectors is currently the subject of intense investigation. In this review, we summarize our current understanding of nematode effectors, with a particular focus on proteinaceous stylet-secreted effectors of sedentary endoparasitic phytonematodes, for which a wealth of information has surfaced in the past 10 yr. We provide an update on the effector repertoires of several of the most economically important genera of phytonematodes and discuss current approaches to dissecting their function. Lastly, we highlight the latest breakthroughs in effector discovery that promise to shed new light on effector diversity and function across the phylum Nematoda.

Review

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