Retromer Contributes to Immunity-Associated Cell Death in Arabidopsis

Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.     

An Atlas of Soybean Small RNAs Identifies Phased siRNAs from Hundreds of Coding Genes

Small RNAs are ubiquitous, versatile repressors and include (1) microRNAs (miRNAs), processed from mRNA forming stem-loops; and (2) small interfering RNAs (siRNAs), the latter derived in plants by a process typically requiring an RNA-dependent RNA polymerase. We constructed and analyzed an expression atlas of soybean (Glycine max) small RNAs, identifying over 500 loci generating 21-nucleotide phased siRNAs (phasiRNAs; from PHAS loci), of which 483 overlapped annotated protein-coding genes. Via the integration of miRNAs with parallel analysis of RNA end (PARE) data, 20 miRNA triggers of 127 PHAS loci were detected. The primary class of PHAS loci (208 or 41% of the total) corresponded to NB-LRR genes; some of these small RNAs preferentially accumulate in nodules. Among the PHAS loci, novel representatives of TAS3 and noncanonical phasing patterns were also observed. A noncoding PHAS locus, triggered by miR4392, accumulated preferentially in anthers; the phasiRNAs are predicted to target transposable elements, with their peak abundance during soybean reproductive development. Thus, phasiRNAs show tremendous diversity in dicots. We identified novel miRNAs and assessed the veracity of soybean miRNAs registered in miRBase, substantially improving the soybean miRNA annotation, facilitating an improvement of miRBase annotations and identifying at high stringency novel miRNAs and their targets.     

The Plant Membrane-Associated REMORIN1.3 Accumulates in Discrete Perihaustorial Domains and Enhances Susceptibility to Phytophthora infestans

Filamentous pathogens such as the oomycete Phytophthora infestans infect plants by developing specialized structures termed haustoria inside the host cells. Haustoria are thought to enable the secretion of effector proteins into the plant cells. Haustorium biogenesis, therefore, is critical for pathogen accommodation in the host tissue. Haustoria are enveloped by a specialized host-derived membrane, the extrahaustorial membrane (EHM), which is distinct from the plant plasma membrane. The mechanisms underlying the biogenesis of the EHM are unknown. Remarkably, several plasma membrane-localized proteins are excluded from the EHM, but the remorin REM1.3 accumulates around P. infestans haustoria. Here, we used overexpression, colocalization with reporter proteins, and superresolution microscopy in cells infected by P. infestans to reveal discrete EHM domains labeled by REM1.3 and the P. infestans effector AVRblb2. Moreover, SYNAPTOTAGMIN1, another previously identified perihaustorial protein, localized to subdomains that are mainly not labeled by REM1.3 and AVRblb2. Functional characterization of REM1.3 revealed that it is a susceptibility factor that promotes infection by P. infestans. This activity, and REM1.3 recruitment to the EHM, require the REM1.3 membrane-binding domain. Our results implicate REM1.3 membrane microdomains in plant susceptibility to an oomycete pathogen.Filamentous plant pathogens, including oomycetes of the genus Phytophthora, downy mildews and white rusts, as well as powdery mildews and rust fungi, are among the most devastating plant pathogens. These biotrophic parasites associate closely with plant cells through specialized infection structures called haustoria. Haustoria are specialized pathogen hyphal structures formed within host cells and enveloped by a perimicrobial membrane called the extrahaustorial membrane (EHM), a key interface between plant pathogens and the host cell. Haustoria are critical for successful parasitic infection by many filamentous plant pathogens and are a signature of the biotrophic lifestyle. In fungi, haustoria function as feeding structures (Voegele et al., 2001). In addition, haustoria are thought to enable the delivery of host-translocated virulence proteins, known as effectors, by both fungal and oomycete pathogens (Catanzariti et al., 2006; Whisson et al., 2007). However, little is known about the molecular mechanisms underlying the biogenesis and function of haustoria and EHM (Kemen and Jones, 2012; Lu et al., 2012).The EHM is thought to be continuous with the host plasma membrane (PM), yet it is a highly specialized membrane compartment that develops only in plant cells that accommodate haustoria (haustoriated cells; Coffey and Wilson, 1983). On the plant side, all eight PM proteins tested by Koh et al. (2005) were excluded from the EHM in Arabidopsis (Arabidopsis thaliana) cells infected with the powdery mildew fungus Golovinomyces cichoracearum. Conversely, the atypical Arabidopsis resistance protein Resistance to Powdery Mildew8.2 (RPW8.2) exclusively localizes to the EHM in this interaction (Wang et al., 2009). Ultrastructure analyses of the Golovinomyces orontii powdery mildew pathosystem revealed that the EHM is asymmetric, thicker and more electron opaque than the PM, and can be highly convoluted around mature haustoria (Micali et al., 2011). More recently, a survey of Arabidopsis and Nicotiana benthamiana plants infected by the oomycete pathogens Hyaloperonospora arabidopsidis and Phytophthora infestans, respectively, revealed that several integral host PM proteins are excluded from the EHM (Lu et al., 2012). Nevertheless, the remorin REM1.3 and the SYNAPTOTAGMIN1 (SYT1) peripheral membrane proteins localized to undetermined subcellular compartments around haustoria in P. infestans-plant interactions (Lu et al., 2012). Whether the differential accumulation of membrane proteins at the EHM is due to interference with the lateral diffusion of proteins from the PM or targeted secretion of specialized vesicles remains unclear (Lu et al., 2012).The subcellular distribution of effectors inside plant cells provides valuable clues about the host cell compartments they modify to promote disease, and effectors have emerged as useful molecular probes for plant cell biology (Whisson et al., 2007; Bozkurt et al., 2012). Heterologous expression of fluorescently tagged effectors in plant cells has been used to determine their subcellular localization in uninfected and infected tissue. This approach has been successful with the RXLR and CRINKLER (CRN) effectors, the two major classes of cytoplasmic (host-translocated) oomycete effectors (Bozkurt et al., 2012). The 49 H. arabidopsidis RXLR effectors studied by Caillaud et al. (2012) localized to the nucleus, the cytoplasm, or various plant membrane compartments. In contrast, CRN effectors from several oomycete species exclusively accumulate in the plant cell nucleus (Schornack et al., 2010; Stam et al., 2013). The P. infestans effectors AVRblb2 and AVR2 accumulate around haustoria when expressed in infected N. benthamiana cells, highlighting the PM and the EHM as important sites for effector activity (Bozkurt et al., 2011; Saunders et al., 2012). These effectors, therefore, can serve as useful probes for plant cell biology to dissect vesicular trafficking and focal immunity, processes that have proved difficult to study using standard genetic approaches (Bozkurt et al., 2011; Win et al., 2012).REM1.3 is one of two plant membrane-associated proteins detected around haustoria during the interaction between P. infestans and the model plant N. benthamiana (Lu et al., 2012). Therefore, we hypothesized that studying REM1.3 should prove useful for understanding the mechanisms governing the function and formation of perihaustorial membranes. REM1.3 belongs to a diverse family of plant-specific proteins containing a Remorin_C domain (PF03763) and has known orthologs in potato (Solanum tuberosum; StREM1.3), tomato (Solanum lycopersicum; SlREM1.2), tobacco (Nicotiana tabacum; NtREM1.2), and Arabidopsis (AtREM1.1–AtREM1.4; Raffaele et al., 2007). Several proteins from the remorin family, including REM1.3, are preferentially associated with membrane rafts, nanometric sterol- and sphingolipid-rich domains in PMs (Pike, 2006; Simons and Gerl, 2010). Indeed, StREM1.3 and NtREM1.2 are highly enriched in detergent-insoluble membranes (DIMs) and form sterol-dependent domains of approximately 75 nm in purified PMs (Mongrand et al., 2004; Shahollari et al., 2004; Raffaele et al., 2009). StREM1.3 directly binds to the cytoplasmic leaflet of the PM through a C-terminal anchor domain (RemCA) that folds into a hairpin of aliphatic α-helices in polar environments (Raffaele et al., 2009; Perraki et al., 2012). StREM1.3 is differentially phosphorylated upon the perception of polygalacturonic acid (Reymond et al., 1996). AtREM1.3 is differentially recruited to DIMs and differentially phosphorylated upon flg22 (for flagellin) peptide perception (Benschop et al., 2007; Keinath et al., 2010; Marín et al., 2012), suggesting a role in plant defense signaling. StREM1.3 and SlREM1.2 prevent Potato virus X spreading by interacting with the Triple Gene Block protein1 (TGBp1) viral movement protein, presumably in plasmodesmata or at the PM (Raffaele et al., 2009; Perraki et al., 2012). AtREM1.2 belongs to protein complexes formed by a negative regulator of immune responses, Resistance to Pseudomonas syringae pv maculicola1 (RPM1)-INTERACTING PROTEIN4, at the PM (Liu et al., 2009). Furthermore, Medicago truncatula MtSYMREM1 is enriched in root cell DIMs (Lefebvre et al., 2007) and localizes to patches at the peribacteroid membrane during symbiosis with Sinorhizobium meliloti (Lefebvre et al., 2010). MtSYMREM1 is important for nodule formation and interacts with the Lysin motif domain–containing receptor-like kinase3 (LYK3) symbiotic receptor (Lefebvre et al., 2010). Multiple lines of evidence, therefore, implicate several remorins in cell surface signaling and the accommodation of microbes during plant-microbe interactions (Raffaele et al., 2007; Jarsch and Ott, 2011; Urbanus and Ott, 2012). Nevertheless, little is known about REM1.3’s molecular function, and its role in immunity against filamentous plant pathogens has not been reported to date.In this study, we analyzed in detail the localization and function of REM1.3 during host colonization by P. infestans. We found that REM1.3 localizes exclusively to the vicinity of the PM and the EHM around noncallosic haustoria. Furthermore, our results suggest that the EHM is likely formed by multiple microdomains. REM1.3 silencing and overexpression experiments demonstrated that it promotes susceptibility to P. infestans in N. benthamiana and tomato. We also show that the REM1.3 membrane anchor domain is required for its localization at the EHM and for the promotion of susceptibility to P. infestans. This work demonstrates the importance of the dynamic reorganization of the PM in response to haustoria-forming pathogens. Our study also revealed that the effector AVRblb2 localizes to remorin-containing host membrane domains at the host-pathogen interface, possibly as a pathogen strategy to facilitate the accommodation of infection structures inside plant cells.     

Evolutionary Relationship of Disease Resistance Genes in Soybean and Arabidopsis Specific for the Pseudomonas syringae Effectors AvrB and AvrRpm1

In Arabidopsis (Arabidopsis thaliana), the Pseudomonas syringae effector proteins AvrB and AvrRpm1 are both detected by the RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) disease resistance (R) protein. By contrast, soybean (Glycine max) can distinguish between these effectors, with AvrB and AvrRpm1 being detected by the Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r R proteins, respectively. We have been using these genes to investigate the evolution of R gene specificity and have previously identified RPM1 and Rpg1b. Here, we report the cloning of Rpg1r, which, like RPM1 and Rpg1b, encodes a coiled-coil (CC)-nucleotide-binding (NB)-leucine-rich repeat (LRR) protein. As previously found for Rpg1b, we determined that Rpg1r is not orthologous with RPM1, indicating that the ability to detect both AvrB and AvrRpm1 evolved independently in soybean and Arabidopsis. The tightly linked soybean Rpg1b and Rpg1r genes share a close evolutionary relationship, with Rpg1b containing a recombination event that combined a NB domain closely related to Rpg1r with CC and LRR domains from a more distantly related CC-NB-LRR gene. Using structural modeling, we mapped polymorphisms between Rpg1b and Rpg1r onto the predicted tertiary structure of Rpg1b, which revealed highly polymorphic surfaces within both the CC and LRR domains. Assessment of chimeras between Rpg1b and Rpg1r using a transient expression system revealed that AvrB versus AvrRpm1 specificity is determined by the C-terminal portion of the LRR domain. The P. syringae effector AvrRpt2, which targets RPM1 INTERACTOR4 (RIN4) proteins in both Arabidopsis and soybean, partially blocked recognition of both AvrB and AvrRpm1 in soybean, suggesting that both Rpg1b and Rpg1r may detect these effectors via modification of a RIN4 homolog.Effector triggered immunity in plants involves highly specific recognition events in which plant resistance (R) proteins detect pathogen effector proteins directly or, alternatively, the modifications that they induce on host proteins (Bonardi et al., 2012). The largest group of R proteins belongs to the nucleotide-binding (NB)-leucine-rich repeat (LRR) family (McHale et al., 2006). The NB-LRR family can be further subdivided based on N-terminal domains into the Toll-Interleukin and R protein (TIR) class and non-TIR-NB-LRR class (McHale et al., 2006). The latter most often contain a coiled-coil (CC) domain at the N terminus. The contributions of the TIR, CC, and LRR domains to R protein specificity, and how new specificities evolve, remain important questions.There are relatively few NB-LRR R proteins characterized to date that are thought to detect pathogen effectors directly; these include Pi-ta from rice (Oryza sativa), L and M variants from flax (Linum usitatissimum), and RESISTANCE TO RALSTONIA SOLANACEARUM1 and RESISTANCE TO PERONOSPORA PARASITICA1 (RPP1) from Arabidopsis (Arabidopsis thaliana; Jia et al., 2000; Deslandes et al., 2003; Dodds et al., 2006; Ueda et al., 2006; Catanzariti et al., 2010; Krasileva et al., 2010). In at least some of these examples, the R genes are found in clusters of NB-LRR paralogs in which multiple recognition specificities are represented (Ellis et al., 1995; Botella et al., 1998) or belong to allelic series (Ellis et al., 1995), arrangements that may promote evolution of recognition specificity via recombination between alleles and paralogs. Interestingly, sequence comparisons and domain swaps involving alleles at the L locus implicate both the LRR and TIR regions as determinants of recognition specificity (Ellis et al., 1999; Luck et al., 2000). Subsequently, domain swaps involving paralogs clustered at the barley (Hordeum vulgare) MILDEW A (MLA) and potato (Solanum tuberosum) Resistance to Potato Virus X (Rx)/Globodera pallida (Gpa) loci have provided additional support for the LRR domain playing a key role in conferring recognition specificity (Ellis et al., 1999; Luck et al., 2000; Shen et al., 2003; Rairdan and Moffett, 2006).Several R proteins are known to detect the presence of pathogen effectors indirectly by monitoring the activity of pathogen effectors within the plant cell. For example, the Arabidopsis RESISTANCE TO PSEUDOMONAS MACULICOLA1 (RPM1) and RESISTANCE TO PSEUDOMONAS SYRINGAE2 (RPS2) R proteins detect modification of the effector target RPM1 INTERACTOR4 (RIN4), while the Arabidopsis RPS5 protein detects modification of the effector target AvrPphB SUSCEPTIBLE1 (Mackey et al., 2002, 2003; Axtell and Staskawicz, 2003; Shao et al., 2003). At least for the well-studied examples in Arabidopsis, R proteins that employ indirect recognition mechanisms are encoded by NB-LRR genes that are not members of large clusters, or allelic series, with variants encoding distinct recognition specificities. Correlated with this genomic structure, such loci are typically relatively stable, with RPM1 and RPS5 existing as presence/absence polymorphisms that have been maintained over long evolutionary periods (Stahl et al., 1999; Tian et al., 2002). Both functional and nonfunctional alleles of RPS2 have been isolated, but only a single recognition specificity has been detected at this locus, despite sequence polymorphisms between alleles (Caicedo et al., 1999).Most likely, specificity for this class of R proteins is determined by a combination of the ability to associate with the host protein targeted by the effector and the ability to detect effector-induced modification of this target. Consistent with this hypothesis, it has been shown that the CC domains from at least some R proteins interact with the host proteins they are monitoring, even in the absence of pathogen effectors, in a prerecognition complex (Mackey et al., 2002; Ade et al., 2007). Hence, evolution of recognition specificity in R proteins that employ indirect recognition mechanisms may involve evolution of both the N-terminal CC and LRR domains.To better understand the evolution and function of R proteins that detect pathogen effectors indirectly, we have been studying two soybean (Glycine max) R genes, with known recognition specificities, that are members of a complex NB-LRR cluster. The R genes involved, Resistance to Pseudomonas glycinea 1b (Rpg1b) and Rpg1r, mediate detection of the Pseudomonas syringae effector proteins AvrB and AvrRpm1, respectively (Staskawicz et al., 1984; Ashfield et al., 1995). We have previously cloned Rpg1b, which is a CC-NB-LRR (CNL) gene that maps to a cluster of R genes effective against a diverse range of pathogens (Ashfield et al., 1998, 2004). Rpg1r is present in the same cluster and maps 0.56 centiMorgans from Rpg1b (Ashfield et al., 1995); however, the evolutionary relationship shared by the two R genes is not known. The cluster is associated with numerous NB-LRR genes, of both the CC and TIR subgroups, spread over more than a megabase of soybean chromosome 13 (Peñuela et al., 2002; Hayes et al., 2004; Innes et al., 2008; Ashfield et al., 2012; Wen et al., 2013). The NB-LRR family in this region is evolving rapidly, with duplications/deletions of paralogs, recombination, and positive selection all playing a role (Ashfield et al., 2012).While soybean can distinguish between AvrB and AvrRpm1, both effectors are detected by a single R protein, RPM1, in Arabidopsis (Bisgrove et al., 1994; Grant et al., 1995). It is known that RPM1 recognizes the effector proteins indirectly by detecting effector-dependent phosphorylation of a second Arabidopsis protein, RIN4 (Mackey et al., 2002; Chung et al., 2011; Liu et al., 2011). The available evidence suggests that a related strategy is employed by soybean, at least for the Rpg1b protein, despite the AvrB recognition specificity having evolved independently in these plant species (Ashfield et al., 2004; Selote and Kachroo, 2010; Selote et al., 2013). Soybean contains four RIN4 homologs (Chen et al., 2010), three of which interact physically with Rpg1b, with two required for full resistance conferred by this R gene (Selote and Kachroo, 2010; Selote et al., 2013). It is not known whether RIN4 homologs are required for Rpg1r function.Here, we report the map-based cloning of the soybean Rpg1r gene. Comparison of the Rpg1r protein to Rpg1b, combined with structural modeling, revealed highly polymorphic surfaces in the CC and LRR domains. Transient expression of chimeric Rpg1 proteins demonstrated that specificity for AvrB versus AvrRpm1 is determined by the C-terminal LRR region. Finally, we provide evidence that Rpg1r, like Rpg1b, detects its corresponding pathogen effector indirectly, most likely by monitoring a RIN4 homolog, indicating convergent evolution of recognition mechanisms in separate plant families.     

The Pseudomonas syringae Effector HopQ1 Promotes Bacterial Virulence and Interacts with Tomato 14-3-3 Proteins in a Phosphorylation-Dependent Manner

A key virulence strategy of bacterial pathogens is the delivery of multiple pathogen effector proteins into host cells during infection. The Hrp outer protein Q (HopQ1) effector from Pseudomonas syringae pv tomato (Pto) strain DC3000 is conserved across multiple bacterial plant pathogens. Here, we investigated the virulence function and host targets of HopQ1 in tomato (Solanum lycopersicum). Transgenic tomato lines expressing dexamethasone-inducible HopQ1 exhibited enhanced disease susceptibility to virulent Pto DC3000, the Pto ΔhrcC mutant, and decreased expression of a pathogen-associated molecular pattern-triggered marker gene after bacterial inoculation. HopQ1-interacting proteins were coimmunoprecipitated and identified by mass spectrometry. HopQ1 can associate with multiple tomato 14-3-3 proteins, including TFT1 and TFT5. HopQ1 is phosphorylated in tomato, and four phosphorylated peptides were identified by mass spectrometry. HopQ1 possesses a conserved mode I 14-3-3 binding motif whose serine-51 residue is phosphorylated in tomato and regulates its association with TFT1 and TFT5. Confocal microscopy and fractionation reveal that HopQ1 exhibits nucleocytoplasmic localization, while HopQ1 dephosphorylation mimics exhibit more pronounced nuclear localization. HopQ1 delivered from Pto DC3000 was found to promote bacterial virulence in the tomato genotype Rio Grande 76R. However, the HopQ1(S51A) mutant delivered from Pto DC3000 was unable to promote pathogen virulence. Taken together, our data demonstrate that HopQ1 enhances bacterial virulence and associates with tomato 14-3-3 proteins in a phosphorylation-dependent manner that influences HopQ1’s subcellular localization and virulence-promoting activities in planta.The ability to detect and mount a defense response against pathogenic microbes is vital for plant survival. Plants rely on both passive and active defenses to ward off microbial pathogens. Physical barriers, such as the cell wall and cuticle, as well as chemical barriers provide a first line of defense against microbial colonization. Unlike animals, plants do not possess a circulating immune system and rely on innate immunity for active defenses against microbial pathogens (Spoel and Dong, 2012). Plants use surface-localized receptors to recognize conserved pathogen-associated molecular patterns (PAMPs), such as bacterial flagellin, resulting in pattern-triggered immunity (PTI; Zipfel et al., 2006). Plants also use primarily intracellular nucleotide-binding domain, Leu-rich repeat containing (NLR) immune receptors to recognize pathogen effectors delivered into host cells during infection (Spoel and Dong, 2012). NLR activation results in effector-triggered immunity (ETI). A signature of ETI is the hypersensitive response (HR), a form of programmed cell death occurring at the site of infection.In order to cause disease and suppress host defense responses, gram-negative bacterial pathogens deliver effector proteins into host cells via the type III secretion system (TTSS). Plant pathogenic bacteria deliver a large number (20–40) of effectors into host cells during infection (Cui et al., 2009). Collectively, effectors are required for bacterial virulence (Lindgren et al., 1986). However, knockouts affecting individual effectors frequently have phenotypes that are subtle, likely due to functional redundancy (Cunnac et al., 2011). Alternatively, individual effectors may play an important role in bacterial survival under conditions that are not typically analyzed in the laboratory or act cooperatively with one another. Progress in understanding individual effectors’ contributions to virulence has been made by generating transgenic plants that express effectors. Multiple effectors have been shown to suppress plant innate immunity and promote bacterial growth when either transiently or stably expressed in plants (Jamir et al., 2004; Guo et al., 2009). Effector expression can also result in avirulent phenotypes when a plant NLR receptor recognizes a cognate effector and mounts an HR. Such an HR phenotype can be used to dissect important effector domains required for plant recognition and enzymatic activity.Elucidating effector targets and enzymatic activity is necessary in order to understand how they act to subvert plant immune responses and can provide elegant insight into biological processes. Significant progress has been made in elucidating the enzymatic activity of a subset of effectors. Some of the most well-characterized effectors come from Pseudomonas syringae pv tomato (Pto), the causal agent of bacterial speck on tomato (Solanum lycopersicum) and Arabidopsis (Arabidopsis thaliana). Multiple effectors can suppress immune responses by directly targeting PAMP receptors (AvrPto and AvrPtoB) or by interfering with downstream signaling processes (AvrB, AvrPphB, and HopAI1; Cui et al., 2009, 2010). The HopU1 effector interferes with RNA metabolism (Fu et al., 2007), and the HopI1 effector targets heat-shock proteins in the plant chloroplast (Jelenska et al., 2010).14-3-3s are conserved eukaryotic proteins that bind a diverse set of phosphorylated client proteins, typically at one of three distinct 14-3-3 binding motifs (Bridges and Moorhead, 2005). There are common recognition motifs for 14-3-3 proteins that contain phosphorylated Ser or Thr residues, but binding to nonphosphorylated ligands and to proteins lacking consensus motifs has been reported (Henriksson et al., 2002; Smith et al., 2011). The 14-3-3 mode I consensus motif is RXXpS/pTX and that of mode II is RXXXpS/pTXP, where X can be any amino acid and p indicates the site of phosphorylation (Smith et al., 2011). 14-3-3 proteins can also bind to the extreme C termini of proteins at the RXXpS/pTX-COOH mode III consensus motif (Smith et al., 2011). Interaction with 14-3-3s can regulate protein activity by influencing client subcellular localization, structure, and protein-protein interactions (Bridges and Moorhead, 2005). Recently, the Xanthomonas campestris XopN effector was shown to target tomato 14-3-3 isoforms, which facilitates its interaction with the tomato atypical receptor kinase1 and suppresses PTI (Kim et al., 2009; Taylor et al., 2012). Other 14-3-3s have also been shown to play a role during plant defense responses. The tomato TFT7 14-3-3 interacts with multiple mitogen-activated protein kinases to positively regulate HR induced by ETI (Oh and Martin, 2011). The Arabidopsis 14-3-3 isoform λ interacts with the RPW8.2 powdery mildew receptor and is required for complete RPW8.2-mediated resistance (Yang et al., 2009).In this study, we investigated the function of the Pto HopQ1 (for Hrp outer protein Q [also known as HopQ1-1]) effector in tomato. HopQ1 is an active effector that is transcribed and translocated via the TTSS (Schechter et al., 2004). HopQ1 induces cell death when expressed in Nicotiana benthamiana and therefore contributes to differences in host range in P. syringae pathovars on Nicotiana spp. (Wei et al., 2007; Ferrante et al., 2009). HopQ1 was also reported to slightly enhance disease symptoms (approximately 0.2 log) and bacterial virulence on bean (Phaseolus vulgaris) when expressed from P. syringae pv tabaci (Ferrante et al., 2009). Here, we generated transgenic tomato plants expressing HopQ1 that exhibited enhanced susceptibility to virulent Pto as well as the Pto ΔhrcC mutant. HopQ1-interacting proteins were identified from tomato using coimmunoprecipitations coupled with mass spectrometry. Multiple 14-3-3 proteins were identified. HopQ1 possesses a 14-3-3 binding motif whose Ser residue is phosphorylated in planta and affects its association with the tomato 14-3-3s TFT1 and TFT5. Mutation of HopQ1’s 14-3-3 binding motif affected its ability to promote bacterial virulence. Taken together, these results indicate that phosphorylation and subsequent interaction with tomato 14-3-3 proteins affect HopQ1’s virulence-promoting activities and subcellular localization.     

The Coronatine Toxin of Pseudomonas syringae Is a Multifunctional Suppressor of Arabidopsis Defense

The phytotoxin coronatine (COR) promotes various aspects of Pseudomonas syringae virulence, including invasion through stomata, growth in the apoplast, and induction of disease symptoms. COR is a structural mimic of active jasmonic acid (JA) conjugates. Known activities of COR are mediated through its binding to the F-box–containing JA coreceptor CORONATINE INSENSITIVE1. By analyzing the interaction of P. syringae mutants with Arabidopsis thaliana mutants, we demonstrate that, in the apoplastic space of Arabidopsis, COR is a multifunctional defense suppressor. COR and the critical P. syringae type III effector HopM1 target distinct signaling steps to suppress callose deposition. In addition to its well-documented ability to suppress salicylic acid (SA) signaling, COR suppresses an SA-independent pathway contributing to callose deposition by reducing accumulation of an indole glucosinolate upstream of the activity of the PEN2 myrosinase. COR also suppresses callose deposition and promotes bacterial growth in coi1 mutant plants, indicating that COR may have multiple targets inside plant cells.     

The Axial Element Protein DESYNAPTIC2 Mediates Meiotic Double-Strand Break Formation and Synaptonemal Complex Assembly in Maize

During meiosis, homologous chromosomes pair and recombine via repair of programmed DNA double-strand breaks (DSBs). DSBs are formed in the context of chromatin loops, which are anchored to the proteinaceous axial element (AE). The AE later serves as a framework to assemble the synaptonemal complex (SC) that provides a transient but tight connection between homologous chromosomes. Here, we showed that DESYNAPTIC2 (DSY2), a coiled-coil protein, mediates DSB formation and is directly involved in SC assembly in maize (Zea mays). The dsy2 mutant exhibits homologous pairing defects, leading to sterility. Analyses revealed that DSB formation and the number of RADIATION SENSITIVE51 (RAD51) foci are largely reduced, and synapsis is completely abolished in dsy2 meiocytes. Super-resolution structured illumination microscopy showed that DSY2 is located on the AE and forms a distinct alternating pattern with the HORMA-domain protein ASYNAPTIC1 (ASY1). In the dsy2 mutant, localization of ASY1 is affected, and loading of the central element ZIPPER1 (ZYP1) is disrupted. Yeast two-hybrid and bimolecular fluorescence complementation experiments further demonstrated that ZYP1 interacts with DSY2 but does not interact with ASY1. Therefore, DSY2, an AE protein, not only mediates DSB formation but also bridges the AE and central element of SC during meiosis.     

Role of Aminoalcoholphosphotransferases 1 and 2 in Phospholipid Homeostasis in Arabidopsis

Aminoalcoholphosphotransferase (AAPT) catalyzes the synthesis of phosphatidylcholine (PC) and phosphotidylethanolamine (PE), which are the most prevalent membrane phospholipids in all eukaryotic cells. Here, we show that suppression of AAPTs results in extensive membrane phospholipid remodeling in Arabidopsis thaliana. Double knockout (KO) mutants that are hemizygous for either aapt1 or aapt2 display impaired pollen and seed development, leading to embryotic lethality of the double KO plants, whereas aapt1 or aapt2 single KO plants show no overt phenotypic alterations. The growth rate and seed yield of AAPT RNA interference (RNAi) plants are greatly reduced. Lipid profiling shows decreased total galactolipid and phospholipid content in aapt1-containing mutants, including aapt1, aapt1/aapt1 aapt2/AAPT2, aapt1/AAPT1 aapt2/aapt2, and AAPT RNAi plants. The level of PC in leaves was unchanged, whereas that of PE was reduced in all AAPT-deficient plants, except aapt2 KO. However, the acyl species of PC was altered, with increased levels of C34 species and decreased C36 species. Conversely, the levels of PE and phosphatidylinositol were decreased in C34 species. In seeds, all AAPT-deficient plants, including aapt2 KO, displayed a decrease in PE. The data show that AAPT1 and AAPT2 are essential to plant vegetative growth and reproduction and have overlapping functions but that AAPT1 contributes more than AAPT2 to PC production in vegetative tissues. The opposite changes in molecular species between PC and PE and unchanged PC level indicate the existence of additional pathways that maintain homeostatic levels of PC, which are crucial for the survival and proper development of plants.     

Salicylic Acid Regulates Arabidopsis Microbial Pattern Receptor Kinase Levels and Signaling

In Arabidopsis thaliana, responses to pathogen-associated molecular patterns (PAMPs) are mediated by cell surface pattern recognition receptors (PRRs) and include the accumulation of reactive oxygen species, callose deposition in the cell wall, and the generation of the signal molecule salicylic acid (SA). SA acts in a positive feedback loop with ACCELERATED CELL DEATH6 (ACD6), a membrane protein that contributes to immunity. This work shows that PRRs associate with and are part of the ACD6/SA feedback loop. ACD6 positively regulates the abundance of several PRRs and affects the responsiveness of plants to two PAMPs. SA accumulation also causes increased levels of PRRs and potentiates the responsiveness of plants to PAMPs. Finally, SA induces PRR- and ACD6-dependent signaling to induce callose deposition independent of the presence of PAMPs. This PAMP-independent effect of SA causes a transient reduction of PRRs and ACD6-dependent reduced responsiveness to PAMPs. Thus, SA has a dynamic effect on the regulation and function of PRRs. Within a few hours, SA signaling promotes defenses and downregulates PRRs, whereas later (within 24 to 48 h) SA signaling upregulates PRRs, and plants are rendered more responsive to PAMPs. These results implicate multiple modes of signaling for PRRs in response to PAMPs and SA.     

MTV1 and MTV4 Encode Plant-Specific ENTH and ARF GAP Proteins That Mediate Clathrin-Dependent Trafficking of Vacuolar Cargo from the Trans-Golgi Network

Many soluble proteins transit through the trans-Golgi network (TGN) and the prevacuolar compartment (PVC) en route to the vacuole, but our mechanistic understanding of this vectorial trafficking step in plants is limited. In particular, it is unknown whether clathrin-coated vesicles (CCVs) participate in this transport step. Through a screen for modified transport to the vacuole (mtv) mutants that secrete the vacuolar protein VAC2, we identified MTV1, which encodes an EPSIN N-TERMINAL HOMOLOGY protein, and MTV4, which encodes the ADP ribosylation factor GTPase-activating protein NEVERSHED/AGD5. MTV1 and NEV/AGD5 have overlapping expression patterns and interact genetically to transport vacuolar cargo and promote plant growth, but they have no apparent roles in protein secretion or endocytosis. MTV1 and NEV/AGD5 colocalize with clathrin at the TGN and are incorporated into CCVs. Importantly, mtv1 nev/agd5 double mutants show altered subcellular distribution of CCV cargo exported from the TGN. Moreover, MTV1 binds clathrin in vitro, and NEV/AGD5 associates in vivo with clathrin, directly linking these proteins to CCV formation. These results indicate that MTV1 and NEV/AGD5 are key effectors for CCV-mediated trafficking of vacuolar proteins from the TGN to the PVC in plants.     

Methylated Cytokinins from the Phytopathogen Rhodococcus fascians Mimic Plant Hormone Activity

Cytokinins (CKs), a class of phytohormones that regulate plant growth and development, are also synthesized by some phytopathogens to disrupt the hormonal balance and to facilitate niche establishment in their hosts. Rhodococcus fascians harbors the fasciation (fas) locus, an operon encoding several genes homologous to CK biosynthesis and metabolism. This pathogen causes unique leafy gall symptoms reminiscent of CK overproduction; however, bacterial CKs have not been clearly correlated with the severe symptoms, and no virulence-associated unique CKs or analogs have been identified. Here, we report the identification of monomethylated N⁶-(∆²-isopentenyl)adenine and dimethylated N⁶-(∆²-isopentenyl)adenine (collectively, methylated cytokinins [MeCKs]) from R. fascians. MeCKs were recognized by a CK receptor and up-regulated type-A ARABIDOPSIS THALIANA RESPONSE REGULATOR genes. Treatment with MeCKs inhibited root growth, a hallmark of CK action, whereas the receptor mutant was insensitive. MeCKs were retained longer in planta than canonical CKs and were poor substrates for a CK oxidase/dehydrogenase, suggesting enhanced biological stability. MeCKs were synthesized by S-adenosyl methionine-dependent methyltransferases (MT1 and MT2) that are present upstream of the fas genes. The best substrate for methylation was isopentenyl diphosphate. MT1 and MT2 catalyzed distinct methylation reactions; only the MT2 product was used by FAS4 to synthesize monomethylated N⁶-(∆²-isopentenyl)adenine. The MT1 product was dimethylated by MT2 and used as a substrate by FAS4 to produce dimethylated N⁶-(∆²-isopentenyl)adenine. Chemically synthesized MeCKs were comparable in activity. Our results strongly suggest that MeCKs function as CK mimics and play a role in this plant-pathogen interaction.The balance of phytohormones, such as cytokinins (CKs) and auxins, is finely controlled to maintain proper plant growth and development but is often disturbed following pathogen infection (Robert-Seilaniantz et al., 2007; Pieterse et al., 2012). As a virulence strategy, many phytopathogens synthesize phytohormones that cause aberrant organogenesis and modulate primary carbon metabolism that ultimately aids disease establishment (Jameson, 2000; Robert-Seilaniantz et al., 2007). For several pathogens, CK production is essential for virulence, and they carry genes for CK biosynthesis in a harbored plasmid (Jameson, 2000). Fungal pathogens employ CKs to form green islands with delayed senescence, whereas bacterial pathogens develop gall structures (Sakakibara et al., 2005; Walters et al., 2008; Giron et al., 2013). Rhodococcus fascians is a gram-positive actinomycete that causes symptoms ranging from leaf deformation to differentiated shooty outgrowths known as leafy galls in more than 150 different plant species (Goethals et al., 2001; Stes et al., 2011). In ornamental plants, such infections reduce their value and contribute to economic losses worldwide (Putnam and Miller, 2007). Leafy gall symptoms are reminiscent of CK overproduction and can be partially induced by exogenous application of CKs (Thimann and Sachs, 1966; Eason et al., 1996). Although several CKs have been isolated from R. fascians culture filtrates, a clear correlation with pathogenesis is lacking partially owing to the low concentration of bacterial CKs (Eason et al., 1996). A synergistic action by a mixture of bacterially produced CKs has been proposed, leading to persistent accumulation of CKs locally (Pertry et al., 2009). Nevertheless, to date, no virulence-associated CK analogs have been identified that could contribute to the infection symptoms.Naturally occurring CKs are adenine derivatives with different side chains at the N6 position. Major plant CKs are N⁶-prenylated adenine derivatives such as N⁶-(Δ²-isopentenyl)adenine (iP), trans-zeatin (tZ), cis-zeatin (cZ), and dihydrozeatin, collectively known as isoprenoid CKs (Sakakibara, 2006). Among them, iP and tZ are the major CKs in Arabidopsis (Arabidopsis thaliana). iP is synthesized by adenosine phosphate-isopentenyl transferase (IPT) using dimethylallyl diphosphate (DMAPP) and adenosine phosphate as substrates (Sakakibara, 2006). tZ is formed by hydroxylation of the trans-end of the prenyl side chain of the iP nucleotide. CK homeostasis is governed by both biosynthesis and catabolism and has an important regulatory role in plant growth (Sakakibara, 2006; Werner et al., 2006). CK oxidase/dehydrogenase (CKX) is responsible for an irreversible reaction cleaving the unsaturated isoprenoid side chain that results in the formation of adenine and the corresponding aldehyde (Werner et al., 2006). In Arabidopsis, CKs are perceived by a subset of sensory His kinases, ARABIDOPSIS HIS KINASE2 (AHK2) to AHK4, which undergo a His-Asp phosphorelay leading to induction of direct target genes including type-A ARABIDOPSIS RESPONSE REGULATOR (ARR) genes (Kieber and Schaller, 2010). This two-component signaling system has been implicated in mediating basal and pathogen-induced plant immunity (Choi et al., 2010; Argueso et al., 2012). For instance, infection of Arabidopsis plants by R. fascians reportedly activates type-A ARR5 expression with increased expression of AHK3 and AHK4, resulting in mitotic cell divisions that arrest the infected leaves in a meristematic state to establish a nutrient-rich niche (Depuydt et al., 2008, 2009; Pertry et al., 2010; Stes et al., 2011). As the infection progresses, IPT genes are switched off, whereas the expression of all CKX genes are strongly induced in symptomatic tissues (Depuydt et al., 2008).The virulence determinant of R. fascians is located within the fasciation (fas) locus, an operon encoding several genes involved in CK metabolism, indicating that CKs are essential for this plant-pathogen interaction (Stes et al., 2011). fas4 encodes IPT that catalyzes the rate-limiting step of CK biosynthesis and is vital for virulence (Stes et al., 2013). Interestingly, two methyltransferase-like genes are present upstream of the fas gene, whose functions have been unknown. Despite the presence of the fas genes in R. fascians, fewer known CKs have been detected compared with other gall-causing pathogens such as Pantoea agglomerans, Agrobacterium tumefaciens, and Pseudomonas savastanoi (Goethals et al., 2001). Further, the leafy gall phenotype is unique, not invoked by any of the above-mentioned pathogens, implying that the virulence of R. fascians might not be due to typical CKs alone (Goethals et al., 2001). R. fascians has long been hypothesized to produce CK analogs using similar or modified substrates (Goethals et al., 2001; Galis et al., 2005; Stes et al., 2011), but no such molecules have been discovered so far. Here, we report the identification and mode of biosynthesis for methylated cytokinins (MeCKs) as hormone mimics from R. fascians. These compounds are synthesized by two methyltransferases and FAS4. Their CK-like activity and higher in planta stability suggest a role for the methylated analogs as CK mimics that foster efficient pathogenesis.