EDS1 complexes are not required for PRR responses and execute TNL-ETI from the nucleus in Nicotiana benthamiana.

Heterodimeric complexes incorporating the lipase-like proteins EDS1 with PAD4 or SAG101 are central hubs in plant innate immunity. EDS1 functions encompass signal relay from TIR domain-containing intracellular NLR-type immune receptors (TNLs) towards RPW8-type helper NLRs (RNLs) and, in Arabidopsis thaliana, bolstering of signaling and resistance mediated by cell-surface pattern recognition receptors (PRRs). Increasing evidence points to the activation of EDS1 complexes by small molecule binding. We used CRISPR/Cas-generated mutant lines and agroinfiltration-based complementation assays to interrogate functions of EDS1 complexes in Nicotiana benthamiana. We did not detect impaired PRR signaling in N. benthamiana lines deficient in EDS1 complexes or RNLs. Intriguingly, in assays monitoring functions of SlEDS1-NbEDS1 complexes in N. benthamiana, mutations within the SlEDS1 catalytic triad could abolish or enhance TNL immunity. Furthermore, nuclear EDS1 accumulation was sufficient for N. benthamiana TNL (Roq1) immunity. Reinforcing PRR signaling in Arabidopsis might be a derived function of the TNL/EDS1 immune sector. Although Solanaceae EDS1 functionally depends on catalytic triad residues in some contexts, our data do not support binding of a TNL-derived small molecule in the triad environment. Whether and how nuclear EDS1 activity connects to membrane pore-forming RNLs remains unknown.

A substrate of the ABC transporter PEN3 stimulates bacterial flagellin (flg22)-induced callose deposition in Arabidopsis thaliana.

Nonhost resistance of Arabidopsis thaliana against Phytophthora infestans, a filamentous eukaryotic microbe and the causal agent of potato late blight, is based on a multilayered defense system. Arabidopsis thaliana controls pathogen entry through the penetration-resistance genes PEN2 and PEN3, encoding an atypical myrosinase and an ABC transporter, respectively, required for synthesis and export of unknown indole compounds. To identify pathogen-elicited leaf surface metabolites and further unravel nonhost resistance in Arabidopsis, we performed untargeted metabolite profiling by incubating a P. infestans zoospore suspension on leaves of WT or pen3 mutant Arabidopsis plants. Among the plant-secreted metabolites, 4-methoxyindol-3-yl-methanol and S-(4-methoxy-indol-3-yl-methyl) cysteine were detected in spore suspensions recollected from WT plants, but at reduced levels from the pen3 mutant plants. In both whole-cell and microsome-based assays, 4-methoxyindol-3-yl-methanol was transported in a PEN3-dependent manner, suggesting that this compound is a PEN3 substrate. The syntheses of both compounds were dependent on functional PEN2 and phytochelatin synthase 1. None of these compounds inhibited mycelial growth of P. infestans in vitro Of note, exogenous application of 4-methoxyindol-3-yl methanol slightly elevated cytosolic Ca2+ levels and enhanced callose deposition in hydathodes of seedlings treated with a bacterial pathogen-associated molecular pattern (PAMP), flagellin (flg22). Loss of flg22-induced callose deposition in leaves of pen3 seedlings was partially reverted by the addition of 4-methoxyindol-3-yl methanol. In conclusion, we have identified a specific indole compound that is a substrate for PEN3 and contributes to the plant defense response against microbial pathogens.

Two Strategies of Pseudomonas syringae to Avoid Recognition of the HopQ1 Effector in Nicotiana Species.

Pseudomonas syringae employs a battery of type three secretion effectors to subvert plant immune responses. In turn, plants have developed receptors that recognize some of the bacterial effectors. Two strain-specific HopQ1 effector variants (for Hrp outer protein Q) from the pathovars phaseolicola 1448A (Pph) and tomato DC3000 (Pto) showed considerable differences in their ability to evoke disease symptoms in Nicotiana benthamiana. Surprisingly, the variants differ by only six amino acids located mostly in the N-terminal disordered region of HopQ1. We found that the presence of serine 87 and leucine 91 renders PtoHopQ1 susceptible to N-terminal processing by plant proteases. Substitutions at these two positions did not strongly affect PtoHopQ1 virulence properties in a susceptible host but they reduced bacterial growth and accelerated onset of cell death in a resistant host, suggesting that N-terminal mutations rendered PtoHopQ1 susceptible to processing in planta and, thus, represent a mechanism of recognition avoidance. Furthermore, we found that co-expression of HopR1, another effector encoded within the same gene cluster masks HopQ1 recognition in a strain-dependent manner. Together, these data suggest that HopQ1 is under high host-pathogen co-evolutionary selection pressure and P. syringae may have evolved differential effector processing or masking as two independent strategies to evade HopQ1 recognition, thus revealing another level of complexity in plant - microbe interactions.

A 1-phytase type III effector interferes with plant hormone signaling.

Most Gram-negative phytopathogenic bacteria inject type III effector (T3E) proteins into plant cells to manipulate signaling pathways to the pathogen's benefit. In resistant plants, specialized immune receptors recognize single T3Es or their biochemical activities, thus halting pathogen ingress. However, molecular function and mode of recognition for most T3Es remains elusive. Here, we show that the Xanthomonas T3E XopH possesses phytase activity, i.e., dephosphorylates phytate (myo-inositol-hexakisphosphate, InsP6), the major phosphate storage compound in plants, which is also involved in pathogen defense. A combination of biochemical approaches, including a new NMR-based method to discriminate inositol polyphosphate enantiomers, identifies XopH as a naturally occurring 1-phytase that dephosphorylates InsP6 at C1. Infection of Nicotiana benthamiana and pepper by Xanthomonas results in a XopH-dependent conversion of InsP6 to InsP5. 1-phytase activity is required for XopH-mediated immunity of plants carrying the Bs7 resistance gene, and for induction of jasmonate- and ethylene-responsive genes in N. benthamiana.

Teaching an old dog new tricks: Suppressing activation of specific mitogen-activated kinases as a potential virulence function of the bacterial AvrRpt2 effector protein.

AvrRpt2 is one of the first Pseudomonas syringae effector proteins demonstrated to be delivered into host cells. It suppresses plant immunity by modulating auxin signaling and cleavage of the membrane-localized defense regulator RIN4. We recently uncovered a novel potential virulence function of AvrRpt2, where it specifically blocked activation of mitogen-activated protein kinases, MPK4 and MPK11, but not of MPK3 and MPK6. Putative AvrRpt2 homologs from different phytopathogens and plant-associated bacteria showed distinct activities with respect to MPK4/11 activation suppression and RIN4 cleavage. Apart from differences in sequence similarity, 3 of the analyzed homologs were apparently "truncated." To examine the role of the AvrRpt2 N-terminus, we modeled the structures of these AvrRpt2 homologs and performed deletion and domain swap experiments. Our results strengthen the finding that RIN4 cleavage is irrelevant for the ability to suppress defense-related MPK4/11 activation and indicate that full protease activity or cleavage specificity is affected by the N-terminus.

Bacterial AvrRpt2-Like Cysteine Proteases Block Activation of the Arabidopsis Mitogen-Activated Protein Kinases, MPK4 and MPK11.

To establish infection, pathogens deliver effectors into host cells to target immune signaling components, including elements of mitogen-activated protein kinase (MPK) cascades. The virulence function of AvrRpt2, one of the first identified Pseudomonas syringae effectors, involves cleavage of the plant defense regulator, RPM1-INTERACTING PROTEIN4 (RIN4), and interference with plant auxin signaling. We show now that AvrRpt2 specifically suppresses the flagellin-induced phosphorylation of Arabidopsis (Arabidopsis thaliana) MPK4 and MPK11 but not MPK3 or MPK6. This inhibition requires the proteolytic activity of AvrRpt2, is associated with reduced expression of some plant defense genes, and correlates with enhanced pathogen infection in AvrRpt2-expressing transgenic plants. Diverse AvrRpt2-like homologs can be found in some phytopathogens, plant-associated and soil bacteria. Employing these putative bacterial AvrRpt2 homologs and inactive AvrRpt2 variants, we can uncouple the inhibition of MPK4/MPK11 activation from the cleavage of RIN4 and related members from the so-called nitrate-induced family as well as from auxin signaling. Thus, this selective suppression of specific mitogen-activated protein kinases is independent of the previously known AvrRpt2 targets and potentially represents a novel virulence function of AvrRpt2.

Agroinfiltration by cytokinin-producing Agrobacterium sp. strain GV3101 primes defense responses in Nicotiana tabacum.

Transient infiltrations in tobacco are commonly used in plant studies, but the host response to different disarmed Agrobacterium strains is not fully understood. The present study shows that pretreatment with disarmed Agrobacterium tumefaciens GV3101 primes the defense response to subsequent infection by Pseudomonas syringae in Nicotiana tabacum. The presence of a trans-zeatin synthase (tzs) gene in strain GV3101 may be partly responsible for the priming response, as the tzs-deficient Agrobacterium sp. strain LBA4404 only weakly imparts such responses. Besides inducing the expression of defense-related genes like PR-1 and NHL10, GV3101 pretreatment increased the expression of tobacco mitogen-activated protein kinase (MAPK) pathway genes like MEK2, WIPK (wound-induced protein kinase), and SIPK (salicylic acid-induced protein kinase). Furthermore, the GV3101 strain showed a stronger effect than the LBA4404 strain in activating phosphorylation of the tobacco MAPK, WIPK and SIPK, which presumably prime the plant immune machinery. Lower doses of exogenously applied cytokinins increased the activation of MAPK, while higher doses decreased the activation, suggesting a balanced level of cytokinins is required to generate defense response in planta. The current study serves as a cautionary warning for plant researchers over the choice of Agrobacterium strains and their possible consequences on subsequent pathogen-related studies.

Unraveling regulation of the small heat shock proteins by the heat shock factor HvHsfB2c in barley: its implications in drought stress response and seed development.

The rapid increase in heat shock proteins upon exposure to damaging stresses and during plant development related to desiccation events reveal their dual importance in plant development and stress tolerance. Genome-wide sequence survey identified 20 non-redundant small heat shock proteins (sHsp) and 22 heat shock factor (Hsf) genes in barley. While all three major classes (A, B, C) of Hsfs are localized in nucleus, the 20 sHsp gene family members are localized in different cell organelles like cytoplasm, mitochondria, plastid and peroxisomes. Hsf and sHsp members are differentially regulated during drought and at different seed developmental stages suggesting the importance of chaperone role under drought as well as seed development. In silico cis-regulatory motif analysis of Hsf promoters showed an enrichment with abscisic acid responsive cis-elements (ABRE), implying regulatory role of ABA in mediating transcriptional response of HvsHsf genes. Gene regulatory network analysis identified HvHsfB2c as potential central regulator of the seed-specific expression of several HvsHsps including 17.5CI sHsp. These results indicate that HvHsfB2c is co-expressed in the central hub of small Hsps and therefore it may be regulating the expression of several HvsHsp subclasses HvHsp16.88-CI, HvHsp17.5-CI and HvHsp17.7-CI. The in vivo relevance of binding specificity of HvHsfB2C transcription factor to HSE-element present in the promoter of HvSHP17.5-CI under heat stress exposure is confirmed by gel shift and LUC-reporter assays. Further, we isolated 477 bp cDNA from barley encoding a 17.5 sHsp polypeptide, which was predominantly upregulated under drought stress treatments and also preferentially expressed in developing seeds. Recombinant HvsHsp17.5-CI protein was expressed in E. coli and purified to homogeneity, which displayed in vitro chaperone activity. The predicted structural model of HvsHsp-17.5-CI protein suggests that the α-crystallin domain is evolutionarily highly conserved.

A pathogen type III effector with a novel E3 ubiquitin ligase architecture.

Type III effectors are virulence factors of Gram-negative bacterial pathogens delivered directly into host cells by the type III secretion nanomachine where they manipulate host cell processes such as the innate immunity and gene expression. Here, we show that the novel type III effector XopL from the model plant pathogen Xanthomonas campestris pv. vesicatoria exhibits E3 ubiquitin ligase activity in vitro and in planta, induces plant cell death and subverts plant immunity. E3 ligase activity is associated with the C-terminal region of XopL, which specifically interacts with plant E2 ubiquitin conjugating enzymes and mediates formation of predominantly K11-linked polyubiquitin chains. The crystal structure of the XopL C-terminal domain revealed a single domain with a novel fold, termed XL-box, not present in any previously characterized E3 ligase. Mutation of amino acids in the central cavity of the XL-box disrupts E3 ligase activity and prevents XopL-induced plant cell death. The lack of cysteine residues in the XL-box suggests the absence of thioester-linked ubiquitin-E3 ligase intermediates and a non-catalytic mechanism for XopL-mediated ubiquitination. The crystal structure of the N-terminal region of XopL confirmed the presence of a leucine-rich repeat (LRR) domain, which may serve as a protein-protein interaction module for ubiquitination target recognition. While the E3 ligase activity is required to provoke plant cell death, suppression of PAMP responses solely depends on the N-terminal LRR domain. Taken together, the unique structural fold of the E3 ubiquitin ligase domain within the Xanthomonas XopL is unprecedented and highlights the variation in bacterial pathogen effectors mimicking this eukaryote-specific activity.

Reduction of divinyl ether-containing polyunsaturated fatty acids in transgenic potato plants.

Oxygenated polyunsaturated fatty acids synthesized via the lipoxygenase pathway play a role in plant responses to pathogen attack. In solanaceous plants, the preferential stimulation of the 9-lipoxygenase pathway in response to pathogen infection leads to the formation of the divinyl ether-containing polyunsaturated fatty acids colneleic and colnelenic acid, as well as hydroxy and trihydroxy polyunsaturated fatty acids. To functionally assess the role of divinyl ethers, transgenic potato plants were generated which express an RNA interference construct directed against the pathogen-inducible 9-divinyl ether synthase. Efficient reduction of 9-divinyl ether synthase transcript accumulation correlated with reduced levels of colneleic and colnelenic acid. However, in response to infection with virulent Phytophthora infestans, the causal agent of late blight disease, no significant differences in pathogen biomass could be detected suggesting that the levels of antimicrobial divinyl ethers are not critical for defense against Phytophthora infestans in a compatible interaction.