A Phytophthora capsici effector suppresses plant immunity via interaction with EDS1

EDS1 (Enhanced Disease Susceptibility 1) plays a crucial role in both effector-triggered immunity activation and plant basal defence. However, whether pathogen effectors can target EDS1 or an EDS1-related pathway to manipulate immunity is rarely reported. In this study, we identified a Phytophthora capsici Avirulence Homolog (Avh) RxLR (Arg-any amino acid-Leu-Arg) effector PcAvh103 that interacts with EDS1. We demonstrated that PcAvh103 can facilitate P. capsici infection and is required for pathogen virulence. Furthermore, genetic evidence showed that PcAvh103 contributes to virulence through targeting EDS1. Finally, PcAvh103 specifically interacts with the lipase domain of EDS1 and can promote the disassociation of EDS1–PAD4 (Phytoalexin Deficient 4) complex in planta. Together, our results revealed that the P. capsici RxLR effector PcAvh103 targets host EDS1 to suppress plant immunity, probably through disrupting the EDS1–PAD4 immune signalling pathway.     

Composting is an effective treatment option for sanitization of Phytophthora ramorum-infected plant material

AIMS: To determine the effects of heat and composting treatments on the viability of the plant pathogen Phytophthora ramorum grown on both artificial and various natural substrates. METHODS AND RESULTS: Phytophthora ramorum was grown on V8 agar, inoculated on bay laurel leaves (Umbellularia californica) and on woody tissues of coast live oak (Quercus agrifolia). Effects on growth, viability and survival were measured as a result of treatment in ovens and compost piles. Direct plating onto PARP medium and pear-baiting techniques were used to determine post-treatment viability. No P. ramorum was recovered at the end of the composting process, regardless of the isolation technique used. By using a PCR assay designed to detect the DNA of P. ramorum, we were able to conclude the pathogen was absent from mature compost and not merely suppressed or dormant. CONCLUSIONS: Some heat and composting treatments eliminate P. ramorum to lower than detectable levels on all substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: Composting is an effective treatment option for sanitization of P. ramorum-infected plant material. Assaying for pathogen viability in compost requires a direct test capable of differentiating between pathogen suppression and pathogen elimination.     

PsHint1, associated with the G‐protein α subunit PsGPA1, is required for the chemotaxis and pathogenicity of Phytophthora sojae

Zoospore chemotaxis to soybean isoflavones is essential in the early stages of infection by the oomycete pathogen Phytophthora sojae. Previously, we have identified a G‐protein α subunit encoded by PsGPA1 which regulates the chemotaxis and pathogenicity of P. sojae. In the present study, we used affinity purification to identify PsGPA1‐interacting proteins, including PsHint1, a histidine triad (HIT) domain‐containing protein orthologous to human HIT nucleotide‐binding protein 1 (HINT1). PsHint1 interacted with both the guanosine triphosphate (GTP)‐ and guanosine diphosphate (GDP)‐bound forms of PsGPA1. An analysis of the gene‐silenced transformants revealed that PsHint1 was involved in the chemotropic response of zoospores to the isoflavone daidzein. During interaction with a susceptible soybean cultivar, PsHint1‐silenced transformants displayed significantly reduced infectious hyphal extension and caused a strong cell death in plants. In addition, the transformants displayed defective cyst germination, forming abnormal germ tubes that were highly branched and exhibited apical swelling. These results suggest that PsHint1 not only regulates chemotaxis by interacting with PsGPA1, but also participates in a Gα‐independent pathway involved in the pathogenicity of P. sojae.     

BON1 interacts with the protein kinases BIR1 and BAK1 in modulation of temperature-dependent plant growth and cell death in Arabidopsis

The Arabidopsis copine gene BON1 encodes a calcium-dependent phospholipid-binding protein involved in plant growth homeostasis and disease resistance. However, the biochemical and molecular mechanisms by which BON1 modulates plant growth and defense responses are not well understood. Here, we show that BON1 interacts physically with the leucine-rich-repeat receptor-like kinases BIR1 (BAK1-interacting receptor-like kinase 1) and pathogen-associated molecular pattern (PAMP) receptor regulator BAK1 in vitro and in vivo. Additionally, bon1 and bir1 mutants exhibit synergistic interaction. While a bir1 null mutant has similar growth and cell-death defects compared with bon1, a bir1 bon1 double mutant displays more severe phenotypes than does the single mutants. The bon1-1 and bir1-1 phenotypes are partially suppressed by overexpression of BIR1 and BON1, respectively. Furthermore, the bir1 phenotype is attenuated by a loss-of-function mutation in the resistance (R) gene SNC1 (Suppressor of npr1-1, constitutive 1), which mediates defense responses in bon1. Intriguingly, BON1 and BIR1 can be phosphorylated by BAK1 in vitro. Our findings suggest that BIR1 functions as a negative regulator of plant resistance and that BON1 and BIR1 might modulate both PAMP- and R protein-triggered immune responses.     

Phytophthora capsici homologue of the cell cycle regulator SDA1 is required for sporangial morphology,mycelial growth and plant infection

SDA1 encodes a highly conserved protein that is widely distributed in eukaryotic organisms. SDA1 is essential for cell cycle progression and organization of the actin cytoskeleton in yeasts and humans. In this study, we identified a Phytophthora capsici orthologue of yeast SDA1, named PcSDA1. In P. capsici, PcSDA1 is strongly expressed in three asexual developmental states (mycelium, sporangia and germinating cysts), as well as late in infection. Silencing or overexpression of PcSDA1 in P. capsici transformants affected the growth of hyphae and sporangiophores, sporangial development, cyst germination and zoospore release. Phalloidin staining confirmed that PcSDA1 is required for organization of the actin cytoskeleton. Moreover, 4′,6‐diamidino‐2‐phenylindole (DAPI) staining and PcSDA1‐green fluorescent protein (GFP) fusions revealed that PcSDA1 is involved in the regulation of nuclear distribution in hyphae and sporangia. Both silenced and overexpression transformants showed severely diminished virulence. Thus, our results suggest that PcSDA1 plays a similar role in the regulation of the actin cytoskeleton and nuclear division in this filamentous organism as in non‐filamentous yeasts and human cells.     

Pan1 is an intrinsically disordered protein with homotypic interactions

The yeast scaffold protein Pan1 contains two EH domains at its N‐terminus, a predicted coiled‐coil central region, and a C‐terminal proline‐rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp³¹² and Trp⁶⁴² are each in an EH domain, Trp⁹⁵⁷ is in the central region, and Trp¹²⁸⁰ is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern‐Volmer constants due to unique electrostatic environments of the tryptophan residues. Time‐resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis. Proteins 2013; 81:1944–1963. © 2013 Wiley Periodicals, Inc.     

Control of the pattern‐recognition receptor EFR by an ER protein complex in plant immunity

In plant innate immunity, the surface‐exposed leucine‐rich repeat receptor kinases EFR and FLS2 mediate recognition of the bacterial pathogen‐associated molecular patterns EF‐Tu and flagellin, respectively. We identified the Arabidopsis stromal‐derived factor‐2 (SDF2) as being required for EFR function, and to a lesser extent FLS2 function. SDF2 resides in an endoplasmic reticulum (ER) protein complex with the Hsp40 ERdj3B and the Hsp70 BiP, which are components of the ER‐quality control (ER‐QC). Loss of SDF2 results in ER retention and degradation of EFR. The differential requirement for ER‐QC components by EFR and FLS2 could be linked to N‐glycosylation mediated by STT3a, a catalytic subunit of the oligosaccharyltransferase complex involved in co‐translational N‐glycosylation. Our results show that the plasma membrane EFR requires the ER complex SDF2–ERdj3B–BiP for its proper accumulation, and provide a demonstration of a physiological requirement for ER‐QC in transmembrane receptor function in plants. They also provide an unexpected differential requirement for ER‐QC and N‐glycosylation components by two closely related receptors.     

Arabidopsis RNA polymerase II C-terminal domain phosphatase-like 1 targets mitogen-activated protein kinase cascades to suppress plant immunity

Mitogen-activated protein kinase (MAPK) cascades play pivotal roles in plant defense against phytopathogens downstream of immune receptor complexes. The amplitude and duration of MAPK activation must be strictly controlled, but the underlying mechanism remains unclear. Here, we identified Arabidopsis CPL1 (C-terminal domain phosphatase-like 1) as a negative regulator of microbe-associated molecular pattern (MAMP)-triggered immunity via a forward-genetic screen. Disruption of CPL1 significantly enhanced plant resistance to Pseudomonas pathogens induced by the bacterial peptide flg22. Furthermore, flg22-induced MPK3/MPK4/MPK6 phosphorylation was dramatically elevated in cpl1 mutants but severely impaired in CPL1 overexpression lines, suggesting that CPL1 might interfere with flg22-induced MAPK activation. Indeed, CPL1 directly interacted with MPK3 and MPK6, as well as the upstream MKK4 and MKK5. A firefly luciferase-based complementation assay indicated that the interaction between MKK4/MKK5 and MPK3/MPK6 was significantly reduced in the presence of CPL1. These results suggest that CPL1 plays a novel regulatory role in suppressing MAMP-induced MAPK cascade activation and MAMP-triggered immunity to bacterial pathogens.     

Functional characterization and immunolocalization of odorant binding protein 1 in the lucerne plant bug, Adelphocoris lineolatus (GOEZE)

In the insect phylum, the relationships between individuals and their environment are often modulated by chemical communication. Odorant binding proteins (OBPs) are widely and robustly expressed in insect olfactory organs and play a key role in chemosensing and transporting hydrophobic odorants across the sensillum lymph to the olfactory receptor neuron. In this study, a novel OBP gene (AlinOBP1) in the lucerne plant bug, Adelphocoris lineolatus was identified, cloned and expressed. Real-time PCR results indicated that the expression level of AlinOBP1 gene differed in each developmental stage (from first instar to adult) and was predominantly expressed in the antennae of adults. The expression level of AlinOBP1 was 1.91 times higher in male antennae than in female antennae. The binding properties of AlinOBP1 with 114 odorants were measured using a fluorescence probe, N-phenyl-1-naphthylamine (1-NPN), with fluorescence competitive binding. The results revealed that AlinOBP1 exhibits high binding abilities with two major putative pheromone components, ethyl butyrate and trans-2-hexenyl butyrate. In addition, it was observed that six volatiles released from cotton, octanal, nonanal, decanal, 2-ethyl-1-hexanol, β-caryophyllene and β-ionone also bind to AlinOBP1. Immunocytochemistry analysis showed that AlinOBP1 was expressed in the sensillum lymph of sensilla trichodica and sensilla basiconca. Our results demonstrate that AlinOBP1 may function as a carrier in the chemoperception of the lucerne plant bug.     

Probing formation of cargo/importin‐α transport complexes in plant cells using a pathogen effector

Importin‐αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin‐α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin‐α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin‐α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co‐opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin‐α paralogs from Arabidopsis thaliana. A crystal structure of the importin‐α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin‐αs expressed in rosette leaves have an almost identical NLS‐binding site. Comparison of the importin‐α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin‐α, sequence variation at the importin‐α NLS‐binding sites and tissue‐specific expression levels of importin‐αs determine formation of cargo/importin‐α transport complexes in plant cells.     

GATA binding protein 3 is correlated with leptin regulation of PPARγ1 in hepatic stellate cells

Accumulating evidence reveals that hormone leptin, mainly produced by adipocyte, plays a unique role in promotion of liver fibrosis. Hepatic stellate cell (HSC) activation is a key step in liver fibrosis and peroxisome‐proliferator activated receptor γ (PPARγ) exerts a crucial role in inhibition of HSC activation. Our previous researches demonstrated that leptin reduced PPARγ1 (a major subtype of PPARγ in HSCs) expression through GATA binding protein 2 (GATA2) binding to a site around ?2323 in PPARγ1 promoter. The present researches aimed to examine the effect of GATA3 on leptin‐induced inhibition of PPARγ1 and elucidate the relationship between GATA3 and GATA2. Gene expressions were analysed by real‐time PCR, western blot, luciferase assay and immunostaining. C57BL/6J ob/ob mouse model of thioacetamide‐induced liver injury was used in vivo. Results demonstrate that leptin significantly induces GATA3 expression in HSCs by multiple signalling pathways including NADPH oxidase pathway. There exist crosstalks between NADPH oxidase pathway and the other pathways. GATA3 can bind to GATA2‐binding site in PPARγ1 promoter and interacts with GATA2, contributing to leptin inhibition of PPARγ1 expression in HSCs. These data demonstrated novel molecular events for leptin inhibition of PPARγ1 expression in HSCs and thus might have potential implications for clarifying the detailed mechanisms underlying liver fibrosis in diseases in which circulating leptin levels are elevated such as non‐alcoholic steatohepatitis in obese patients.     

The plant nuclear envelope protein MAF1 has an additional location at the Golgi and binds to a novel Golgi-associated coiled-coil protein

Tomato MAF1 (LeMAF1) is a plant-specific, nuclear envelope (NE)-associated protein. It is the founding member of a group of WPP domain-containing, NE-associated proteins. This group includes the Arabidopsis WPP family, which is involved in cell division, as well as plant RanGAPs. In addition to its NE localization, LeMAF1 accumulates in speckles in the cytoplasm. Here, we show that the LeMAF1-containing speckles are components of the Golgi apparatus. A novel tomato coiled-coil protein was identified that specifically binds to LeMAF1. Tomato WPP domain-associated protein (LeWAP) interacts in yeast and in vitro through its coiled-coil domain with several WPP-domain containing proteins, including AtRanGAP1 and the WPP family (LeMAF, WPP1 and WPP2). Like LeMAF1, LeWAP is localized at the Golgi. Moreover, we present data showing that Arabidopsis WAP is necessary for the existence of a multi-protein complex containing WPP2. Electronic Supplementary Material Supplementary material is available for this article at .     

Sorting nexin 17, a non-self-assembling and a PtdIns(3)P high class affinity protein, interacts with the cerebral cavernous malformation related protein KRIT1

The mammalian sorting nexin (SNX) proteins are involved in the endocytosis and the sorting machinery of transmembrane proteins. Additionally to the family defining phox homology (PX) domain, SNX17 is the only member with a truncated FERM (4.1, ezrin, radixin, and moesin) domain and a unique C-terminal region (together designated as FC unit). By gel filtration and lipid overlay assays we show that SNX17 is a non-self-assembling and a PtdIns(3)P high class affinity protein. A SNX17 affinity to any other phosphoinositides was not detected. By yeast two-hybrid- and GST-trapping assays we identified KRIT1 (krev1 interaction trapped 1) as a new specific interaction partner of the FC unit of SNX17. KRIT1 binds SNX17 by its N-terminal region like the known interaction partner ICAP1alpha (integrin cytoplasmic domain-associated protein-1). The interaction was also detected in HEK 293 cells transiently expressing GFP-tagged KRIT1 and Xpress-tagged SNX17. KRIT1 mutations cause cerebral cavernous malformation (CCM1). Our finding suggests a SNX17 involvement in the indicated KRIT1 function in cell adhesion processes by integrin signaling.     

Induced alignment and measurement of dipolar couplings of an SH2 domain through direct binding with filamentous phage

Large residual ¹⁵N-¹H dipolar couplings have been measured in a Src homology II domain aligned at Pf1 bacteriophage concentrations an order of magnitude lower than used for induction of a similar degree of alignment of nucleic acids and highly acidic proteins. An increase in ¹ H and ¹⁵N protein linewidths and a decrease in T₂ and T₁ relaxation time constants implicates a binding interaction between the protein and phage as the mechanism of alignment. However, the associated increased linewidth does not preclude the accurate measurement of large dipolar couplings in the aligned protein. A good correlation is observed between measured dipolar couplings and predicted values based on the high resolution NMR structure of the SH2 domain. The observation of binding-induced protein alignment promises to broaden the scope of alignment techniques by extending their applicability to proteins that are able to interact weakly with the alignment medium.     

The Arabidopsis FLC protein interacts directly in vivo with SOC1 and FT chromatin and is part of a high-molecular-weight protein complex

The Arabidopsis Flowering Locus C (FLC) protein is a repressor of flowering regulated by genes in the autonomous and vernalization pathways. Previous genetic and transgenic data have suggested that FLC acts by repressing expression of the floral integrator genes SOC1 and FT. We have taken an in vivo approach to determine whether the FLC protein interacts directly with potential DNA targets. Using chromatin immunoprecipitation, we have shown that FLC binds to a region of the first intron of FT that contains a putative CArG box, and have confirmed that FLC binds to a CArG box in the promoter of the SOC1 gene. MADS box proteins are thought to bind their DNA targets as dimers or higher-order multimers. We have shown that FLC is a component of a multimeric protein complex in vivo and that more than one FLC polypeptides can be present in the complex.