A Bacterial Effector Mimics a Host HSP90 Client to Undermine Immunity.

The molecular chaperone HSP90 facilitates the folding of several client proteins, including innate immune receptors and protein kinases. HSP90 is an essential component of plant and animal immunity, yet pathogenic strategies that directly target the chaperone have not been described. Here, we identify the HopBF1 family of bacterial effectors as eukaryotic-specific HSP90 protein kinases. HopBF1 adopts a minimal protein kinase fold that is recognized by HSP90 as a host client. As a result, HopBF1 phosphorylates HSP90 to completely inhibit the chaperone's ATPase activity. We demonstrate that phosphorylation of HSP90 prevents activation of immune receptors that trigger the hypersensitive response in plants. Consequently, HopBF1-dependent phosphorylation of HSP90 is sufficient to induce severe disease symptoms in plants infected with the bacterial pathogen, Pseudomonas syringae. Collectively, our results uncover a family of bacterial effector kinases with toxin-like properties and reveal a previously unrecognized betrayal mechanism by which bacterial pathogens modulate host immunity.

The Rysto immune receptor recognises a broadly conserved feature of potyviral coat proteins.

Knowledge of the immune mechanisms responsible for viral recognition is critical for understanding durable disease resistance and successful crop protection. We determined how potato virus Y (PVY) coat protein (CP) is recognised by Rysto , a TNL immune receptor. We applied structural modelling, site-directed mutagenesis, transient overexpression, co-immunoprecipitation, infection assays and physiological cell death marker measurements to investigate the mechanism of Rysto -CP interaction. Rysto associates directly with PVY CP in planta that is conditioned by the presence of a CP central 149 amino acids domain. Each deletion that affects the CP core region impairs the ability of Rysto to trigger defence. Point mutations in the amino acid residues Ser125 , Arg157 , and Asp201 of the conserved RNA-binding pocket of potyviral CP reduce or abolish Rysto binding and Rysto -dependent responses, demonstrating that appropriate folding of the CP core is crucial for Rysto -mediated recognition. Rysto recognises the CPs of at least 10 crop-damaging viruses that share a similar core region. It confers immunity to plum pox virus and turnip mosaic virus in both Solanaceae and Brassicaceae systems, demonstrating potential utility in engineering virus resistance in various crops. Our findings shed new light on how R proteins detect different viruses by sensing conserved structural patterns.

Arabidopsis annexin 5 is involved in maintenance of pollen membrane integrity and permeability.

In Arabidopsis, a dry stigma surface enables a gradual hydration of pollen grains by a controlled release of water. Occasionally the grains may be exposed to extreme precipitations that cause rapid water influx and swelling, eventually leading to pollen membrane rupture. In metazoans, calcium- and phospholipid-binding proteins, referred to as annexins, participate in the repair of plasma membrane damages. It remains unclear, however, how this process is conducted in plants. Here, we examined whether plant annexin 5 (ANN5), the most abundant member of the annexin family in pollen, is involved in the restoration of pollen membrane integrity. We analyzed the cellular dynamics of ANN5 in pollen grains undergoing hydration in favorable or stress conditions. We observed a transient association of ANN5 with the pollen membrane during in vitro hydration that did not occur in the pollen grains being hydrated on the stigma. To simulate a rainfall, we performed spraying of the pollinated stigma with deionized water that induced ANN5 accumulation at the pollen membrane. Interestingly, calcium or magnesium application affected pollen membrane properties differently, causing rupture or shrinkage of pollen membrane, respectively. Both treatments, however, induced ANN5 recruitment to the pollen membrane. Our data suggest a model in which ANN5 is involved in the maintenance of membrane integrity in pollen grains exposed to osmotic or ionic imbalances.

Precision transcriptomics of viral foci reveals the spatial regulation of immune-signaling genes and identifies RBOHD as an important player in the incompatible interaction between potato virus Y and potato.

Whereas the activation of resistance (R) proteins has been intensively studied, the downstream signaling mechanisms leading to the restriction of the pathogen remain mostly unknown. We studied the immunity network response conditioned by the potato Ny-1 gene against potato virus Y. We analyzed the processes in the cell death zone and surrounding tissue on the biochemical and gene expression levels in order to reveal the spatiotemporal regulation of the immune response. We show that the transcriptional response in the cell death zone and surrounding tissue is dependent on salicylic acid (SA). For some genes the spatiotemporal regulation is completely lost in the SA-deficient line, whereas other genes show a different response, indicating multiple connections between hormonal signaling modules. The induction of NADPH oxidase RBOHD expression occurs specifically on the lesion border during the resistance response. In plants with silenced RBOHD, the functionality of the resistance response is perturbed and the spread of the virus is not arrested at the site of infection. RBOHD is required for the spatial accumulation of SA, and conversely RBOHD is under the transcriptional regulation of SA. Using spatially resolved RNA-seq, we also identified spatial regulation of an UDP-glucosyltransferase, another component in feedback activation of SA biosynthesis, thus deciphering a novel aspect of resistance signaling.

Extreme resistance to Potato virus Y in potato carrying the Rysto gene is mediated by a TIR-NLR immune receptor.

Potato virus Y (PVY) is a major potato (Solanum tuberosum L.) pathogen that causes severe annual crop losses worth billions of dollars worldwide. PVY is transmitted by aphids, and successful control of virus transmission requires the extensive use of environmentally damaging insecticides to reduce vector populations. Rysto , from the wild relative S. stoloniferum, confers extreme resistance (ER) to PVY and related viruses and is a valuable trait that is widely employed in potato resistance breeding programmes. Rysto was previously mapped to a region of potato chromosome XII, but the specific gene has not been identified to date. In this study, we isolated Rysto using resistance gene enrichment sequencing (RenSeq) and PacBio SMRT (Pacific Biosciences single-molecule real-time sequencing). Rysto was found to encode a nucleotide-binding leucine-rich repeat (NLR) protein with an N-terminal TIR domain and was sufficient for PVY perception and ER in transgenic potato plants. Rysto -dependent extreme resistance was temperature-independent and requires EDS1 and NRG1 proteins. Rysto may prove valuable for creating PVY-resistant cultivars of potato and other Solanaceae crops.

Nucleus- and plastid-targeted annexin 5 promotes reproductive development in Arabidopsis and is essential for pollen and embryo formation.

BACKGROUND: Pollen development is a strictly controlled post-meiotic process during which microspores differentiate into microgametophytes and profound structural and functional changes occur in organelles. Annexin 5 is a calcium- and lipid-binding protein that is highly expressed in pollen grains and regulates pollen development and physiology. To gain further insights into the role of ANN5 in Arabidopsis development, we performed detailed phenotypic characterization of Arabidopsis plants with modified ANN5 levels. In addition, interaction partners and subcellular localization of ANN5 were analyzed to investigate potential functions of ANN5 at cellular level. RESULTS: Here, we report that RNAi-mediated suppression of ANN5 results in formation of smaller pollen grains, enhanced pollen lethality, and delayed pollen tube growth. ANN5 RNAi knockdown plants also displayed aberrant development during the transition from the vegetative to generative phase and during embryogenesis, reflected by delayed bolting time and reduced embryo size, respectively. At the subcellular level, ANN5 was delivered to the nucleus, nucleolus, and cytoplasm, and was frequently localized in plastid nucleoids, suggesting a likely role in interorganellar communication. Furthermore, ANN5-YFP co-immunoprecipitated with RABE1b, a putative GTPase, and interaction in planta was confirmed in plastidial nucleoids using FLIM-FRET analysis. CONCLUSIONS: Our findings let us to propose that ANN5 influences basal cell homeostasis via modulation of plastid activity during pollen maturation. We hypothesize that the role of ANN5 is to orchestrate the plastidial and nuclear genome activities via protein-protein interactions however not only in maturing pollen but also during the transition from the vegetative to the generative growth and seed development.

Stress-Related Gene Expression Reflects Morphophysiological Responses to Water Deficit.

Acclimation to water deficit (WD) enables plants to maintain growth under unfavorable environmental conditions, although the mechanisms are not completely understood. In this study, the natural variation of long-term acclimation to moderate and severe soil WD was investigated in 18 Arabidopsis (Arabidopsis thaliana) accessions using PHENOPSIS, an automated phenotyping platform. Soil water content was adjusted at an early stage of plant development and maintained at a constant level until reproductive age was achieved. The accessions were selected based on the expression levels of ANNEXIN1, a drought-related marker. Severe WD conditions had a greater effect on most of the measured morphophysiological traits than moderate WD conditions. Multivariate analyses indicated that trait responses associated with plant size and water management drove most of the variation. Accessions with similar responses at these two levels were grouped in clusters that displayed different response strategies to WD The expression levels of selected stress-response genes revealed large natural variation under WD conditions. Responses of morphophysiological traits, such as projected rosette area, transpiration rate, and rosette water content, were correlated with changes in the expression of stress-related genes, such as NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 and N-MYC DOWNREGULATED-LIKE1 (NDL1), in response to WD Interestingly, the morphophysiological acclimation response to WD also was reflected in the gene expression levels (most notably those of NDL1, CHALCONE SYNTHASE, and MYB DOMAIN PROTEIN44) in plants cultivated under well-watered conditions. Our results may lead to the development of biomarkers and predictors of plant morphophysiological responses based on gene expression patterns.

Phosphorylation of HopQ1, a type III effector from Pseudomonas syringae, creates a binding site for host 14-3-3 proteins.

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.

Structures of an active-site mutant of a plant 1,3-ß-glucanase in complex with oligosaccharide products of hydrolysis.

Plant endo-1,3-ß-glucanases are involved in important physiological processes such as defence mechanisms, cell division and flowering. They hydrolyze (1→3)-ß-glucans, with very limited activity towards mixed (1→3,1→4)-ß-glucans and branched (1→3,1→6)-ß-glucans. Here, crystal structures of the potato (Solanum tuberosum) endo-1,3-ß-glucanase GLUB20-2 with the nucleophilic Glu259 residue substituted by alanine (E259A) are reported. Despite this active-site mutation, the protein retained residual endoglucanase activity and when incubated in the crystallization buffer with a linear hexameric substrate derived from (1→3)-ß-glucan (laminarahexose) cleaved it in two different ways, generating trisaccharides and tetrasaccharides, as confirmed by mass spectrometry. The trisaccharide (laminaratriose) shows higher binding affinity and was found to fully occupy the -1, -2 and -3 sites of the active-site cleft, even at a low molar excess of the substrate. At elevated substrate concentration the tetrasaccharide molecule (laminaratetrose) also occupies the active site, spanning the opposite sites +1, +2, +3 and +4 of the cleft. These are the first crystal structures of a plant glycoside hydrolase family 17 (GH17) member to reveal the protein-saccharide interactions and were determined at resolutions of 1.68 and 1.55 Å, respectively. The geometry of the active-site cleft clearly precludes any (1→4)-ß-glucan topology at the subsites from -3 to +4 and could possibly accommodate ß-1,6-branching only at subsites +1 and +2. The glucose units at subsites -1 and -2 interact with highly conserved protein residues. In contrast, subsites -3, +3 and +4 are variable, suggesting that the mode of glucose binding at these sites may vary between different plant endo-1,3-ß-glucanases. Low substrate affinity is observed at subsites +1 and +2, as manifested by disorder of the glycosyl units there.

Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato.

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants.