Effector Proteins Research Articles

Effector Protein Serine Carboxypeptidase FgSCP Is Essential for Full Virulence in Fusarium graminearum and Is Involved in Modulating Plant Immune Responses.

Fusarium head blight caused by Fusarium graminearum is a significant pathogen affecting wheat crops. During the infection process, effector proteins are secreted to modulate plant immunity and promote infection. The toxin deoxynivalenol is produced in infected wheat grains, posing a threat to human and animal health. Serine carboxypeptidases (SCPs) belong to the α/β hydrolase family of proteases and are widely distributed in plant and fungal vacuoles, as well as animal lysosomes. Research on SCPs mainly focuses on the isolation, purification, and production of a small number of fungi. The role of SCPs in plant secretion, growth and development, and stress resistance has also been extensively studied. However, their functions in F. graminearum, a fungal pathogen, remain relatively unknown. In this study, the biological functions of the FgSCP gene in F. graminearum were investigated. The study revealed that mutations in FgSCP affected the nutritional growth, sexual reproduction, and stress tolerance of F. graminearum. Furthermore, the deletion of FgSCP resulted in reduced pathogenicity and hindered the biosynthesis of deoxynivalenol. The upregulation of FgSCP expression 3 days after infection indicated its involvement in host invasion, possibly acting as a "smokescreen" to deceive the host and suppress the expression of host defensive genes. Subsequently, we confirmed the secretion ability of FgSCP and its ability to inhibit the cell death induced by INF1 in Nicotiana benthamiana cells, indicating its potential role as an effector protein in suppressing plant immune responses and promoting infection. In summary, we have identified FgSCP as an essential effector protein in F. graminearum, playing critical roles in growth, virulence, secondary metabolism, and host invasion.

The Soybean Cyst Nematode Effector Cysteine Protease 1 (CPR1) Targets a Mitochondrial Soybean Branched-Chain Amino Acid Aminotransferase (GmBCAT1).

The soybean cyst nematode (SCN; Heterodera glycines) facilitates infection by secreting a repertoire of effector proteins into host cells to establish a permanent feeding site composed of a syncytium of root cells. Among the diverse proteins secreted by the nematode, we were specifically interested in identifying proteases to pursue our goal of engineering decoy substrates that elicit an immune response when cleaved by an SCN protease. We identified a cysteine protease that we named Cysteine Protease 1 (CPR1), which was predicted to be a secreted effector based on transcriptomic data obtained from SCN esophageal gland cells, presence of a signal peptide, and lack of transmembrane domains. CPR1 is conserved in all isolates of SCN sequenced to date, suggesting it is critical for virulence. Transient expression of CPR1 in Nicotiana benthamiana leaves suppressed cell death induced by a constitutively active nucleotide binding leucine-rich repeat protein, RPS5, indicating that CPR1 inhibits effector-triggered immunity. CPR1 localizes in part to the mitochondria when expressed in planta. Proximity-based labeling in transgenic soybean roots, co-immunoprecipitation, and cleavage assays identified a branched-chain amino acid aminotransferase from soybean (GmBCAT1) as a substrate of CPR1. Consistent with this, GmBCAT1 also localizes to mitochondria. Silencing of the CPR1 transcript in the nematode reduced penetration frequency in soybean roots while the expression of CPR1 in soybean roots enhanced susceptibility. Our data demonstrates that CPR1 is a conserved effector protease with a direct target in soybean roots, highlighting it as a promising candidate for decoy engineering.

Capturing RAS oligomerization on a membrane

RAS GTPases associate with the biological membrane where they function as molecular switches to regulate cell growth. Recent studies indicate that RAS proteins oligomerize on membranes, and disrupting these assemblies represents an alternative therapeutic strategy. However, conflicting reports on RAS assemblies, ranging in size from dimers to nanoclusters, have brought to the fore key questions regarding the stoichiometry and parameters that influence oligomerization. Here, we probe three isoforms of RAS [Kirsten Rat Sarcoma viral oncogene (KRAS), Harvey Rat Sarcoma viral oncogene (HRAS), and Neuroblastoma oncogene (NRAS)] directly from membranes using mass spectrometry. We show that KRAS on membranes in the inactive state (GDP-bound) is monomeric but forms dimers in the active state (GTP-bound). We demonstrate that the small molecule BI2852 can induce dimerization of KRAS, whereas the binding of effector proteins disrupts dimerization. We also show that RAS dimerization is dependent on lipid composition and reveal that oligomerization of NRAS is regulated by palmitoylation. By monitoring the intrinsic GTPase activity of RAS, we capture the emergence of a dimer containing either mixed nucleotides or GDP on membranes. We find that the interaction of RAS with the catalytic domain of Son of Sevenless (SOScat) is influenced by membrane composition. We also capture the activation and monomer to dimer conversion of KRAS by SOScat. These results not only reveal the stoichiometry of RAS assemblies on membranes but also uncover the impact of critical factors on oligomerization, encompassing regulation by nucleotides, lipids, and palmitoylation.

Hippo pathway effectors YAP, TAZ and TEAD are associated with EMT master regulators ZEB, Snail and with aggressive phenotype in phyllodes breast tumors

BackgroundPhyllodes tumors (PTs) of the breast are uncommon fibroepithelial neoplasms that tend to recur locally and may have metastatic potential. Their pathogenesis is poorly understood. Hippo signaling pathway plays an essential role in organ size control, tumor suppression, tissue regeneration and stem cell self-renewal. Hippo signaling dysfunction has been implicated in cancer. Recent evidence suggests that there is cross-talk between the Hippo signaling key proteins YAP/TAZ and the epithelial-mesenchymal transition (EMT) master regulators Snail and ZEB. In this study we aimed to investigate the expression of Hippo signaling pathway components and EMT regulators in PTs, in relation to tumor grade. MethodsExpression of Hippo signaling effector proteins YAP, TAZ and their DNA binding partner TEAD was evaluated by immunohistochemistry in paraffin-embedded tissue specimens from 86 human phyllodes breast tumors (45 benign, 21 borderline, 20 malignant), in comparison with tumor grade and with the expression of EMT-related transcription factors ZEB and Snail. ResultsNuclear immunopositivity for YAP, TAZ and TEAD was detected in both stromal and epithelial cells in PTs and was significantly higher in high grade tumors. Interestingly, there was a significant correlation between the expression of YAP, TAZ, TEAD and the expression of ZEB and SNAIL. ConclusionsOur results originally implicate Hippo signaling pathway in PTs pathogenesis and suggest that an interaction between Hippo signaling key components and EMT regulators may promote the malignant features of PTs.

Wheat Leaf Rust Fungus Effector Protein Pt1641 Is Avirulent to TcLr1.

Wheat leaf rust fungus is an obligate parasitic fungus that can absorb nutrients from its host plant through haustoria and secrete effector proteins into host cells. The effector proteins are crucial factors for pathogenesis as well as targets for host disease resistance protein recognition. Exploring the role of effector proteins in the pathogenic process of Puccinia triticina Eriks. (Pt) is of great significance for unraveling its pathogenic mechanisms. We previously found that a cysteine-rich effector protein, Pt1641, is highly expressed during the interaction between wheat and Pt, but its specific role in pathogenesis remains unclear. Therefore, this study employed techniques such as heterologous expression, qRT-PCR analysis, and host-induced gene silencing (HIGS) to investigate the role of Pt1641 in the pathogenic process of Pt. The results indicate that Pt1641 is an effector protein with a secretory function and can inhibit BAX-induced programmed cell death in Nicotiana benthamiana. qRT-PCR analyses showed that expression levels of Pt1641 were different during the interaction between the high-virulence strain THTT and low-virulence strains FGD and Thatcher, respectively. The highest expression level in the low-virulence strain FGD was four times that of the high-virulence strain THTT. The overexpression of Pt1641 in wheat near-isogenic line TcLr1 induced callose deposition and H2O2 production on TcLr1. After silencing Pt1641 in the Pt low-virulence strain FGD on wheat near-isogenic line TcLr1, the pathogenic phenotype of Pt physiological race FGD on TcLr1 changed from ";" to "3", indicating that Pt1641 plays a non-toxic function in the pathogenicity of FGD to TcLr1. This study helps to reveal the pathogenic mechanism of wheat leaf rust and provides important guidance for the mining and application of Pt avirulent genes.

S-acylation of Ca2+ transport proteins in cancer.

Alterations in cellular calcium (Ca2+) signals have been causally associated with the development and progression of human cancers. Cellular Ca2+ signals are generated by channels, pumps, and exchangers that move Ca2+ ions across membranes and are decoded by effector proteins in the cytosol or in organelles. S-acylation, the reversible addition of 16-carbon fatty acids to proteins, modulates the activity of Ca2+ transporters by altering their affinity for lipids, and enzymes mediating this reversible post-translational modification have also been linked to several types of cancers. Here, we compile studies reporting an association between Ca2+ transporters or S-acylation enzymes with specific cancers, as well as studies reporting or predicting the S-acylation of Ca2+ transporters. We then discuss the potential role of S-acylation in the oncogenic potential of a subset of Ca2+ transport proteins involved in cancer.

Coxiella burnetii protein CBU2016 supports CCV expansion.

Coxiella burnetii is a globally distributed obligate intracellular pathogen. Although often asymptomatic, infections can cause acute Q fever with influenza-like symptoms and/or severe chronic Q fever. Coxiella burnetii develops a unique replicative niche within host cells called the Coxiella-containing vacuole (CCV), facilitated by the Dot/Icm type IV secretion system translocating a cohort of bacterial effector proteins into the host. The role of some effectors has been elucidated; however, the actions of the majority remain enigmatic and the list of true effectors is disputable. This study examined CBU2016, a unique C. burnetii protein previously designated as an effector with a role in infection. We were unable to validate CBU2016 as a translocated effector protein. Employing targeted knock-out and complemented strains, we found that the loss of CBU2016 did not cause a replication defect within Hela, THP-1, J774, or iBMDM cells or in axenic media, nor did it affect the pathogenicity of C. burnetii in the Galleria mellonella infection model. The absence of CBU2016 did, however, result in a consistent decrease in the size of CCVs in HeLa cells. These results suggest that although CBU2016 may not be a Dot/Icm effector, it is still able to influence the host environment during infection.

Genome scans for selection signatures identify candidate virulence genes for adaptation of the soybean cyst nematode to host resistance.

Plant pathogens are constantly under selection pressure for host resistance adaptation. Soybean cyst nematode (SCN, Heterodera glycines) is a major pest of soybean primarily managed through resistant cultivars; however, SCN populations have evolved virulence in response to selection pressures driven by repeated monoculture of the same genetic resistance. Resistance to SCN is mediated by multiple epistatic interactions between Rhg (for resistance to H. glycines) genes. However, the identity of SCN virulence genes that confer the ability to overcome resistance remains unknown. To identify candidate genomic regions showing signatures of selection for increased virulence, we conducted whole genome resequencing of pooled individuals (Pool-Seq) from two pairs of SCN populations adapted on soybeans with Peking-type (rhg1-a, rhg2, and Rhg4) resistance. Population differentiation and principal component analysis-based approaches identified approximately 0.72-0.79 million SNPs, the frequency of which showed potential selection signatures across multiple genomic regions. Chromosomes 3 and 6 between population pairs showed the greatest density of outlier SNPs with high population differentiation. Conducting multiple outlier detection tests to identify overlapping SNPs resulted in a total of 966 significantly differentiated SNPs, of which 285 exon SNPs were mapped to 97 genes. Of these, six genes encoded members of known stylet-secreted effector protein families potentially involved in host defence modulation including venom-allergen-like, annexin, glutathione synthetase, SPRYSEC, chitinase, and CLE effector proteins. Further functional analysis of identified candidate genes will provide new insights into the genetic mechanisms by which SCN overcomes soybean resistance and inform the development of molecular markers for rapidly screening the virulence profile of an SCN-infested field.

Commensal Yeast Promotes Salmonella Typhimurium Virulence.

Enteric pathogens engage in complex interactions with the host and the resident microbiota to establish gut colonization. Although mechanistic interactions between enteric pathogens and bacterial commensals have been extensively studied, whether and how commensal fungi affect pathogenesis of enteric infections remains largely unknown. Here we show that colonization with the common human gut commensal fungus Candida albicans worsened infections with the enteric pathogen Salmonella enterica serovar Typhimurium. Presence of C. albicans in the mouse gut increased Salmonella cecum colonization and systemic dissemination. We investigated the underlying mechanism and found that Salmonella binds to C. albicans via Type 1 fimbriae and uses its Type 3 Secretion System (T3SS) to deliver effector proteins into C. albicans . A specific effector, SopB, was sufficient to manipulate C. albicans metabolism, triggering increased arginine biosynthesis in C. albicans and the release of millimolar amounts of arginine into the extracellular environment. The released arginine, in turn, induced T3SS expression in Salmonella , increasing its invasion of epithelial cells. C. albicans deficient in arginine production was unable to increase Salmonella virulence in vitro or in vivo . In addition to modulating pathogen invasion, arginine also directly influenced the host response to infection. Arginine-producing C. albicans dampened the inflammatory response during Salmonella infection, whereas C. albicans deficient in arginine production did not. Arginine supplementation in the absence of C. albicans increased the systemic spread of Salmonella and decreased the inflammatory response, phenocopying the presence of C. albicans . In summary, we identified C. albicans colonization as a susceptibility factor for disseminated Salmonella infection, and arginine as a central metabolite in the cross-kingdom interaction between fungi, bacteria, and host.

Fusarium graminearum effector FgEC1 targets wheat TaGF14b protein to suppress TaRBOHD-mediated ROS production and promote infection.

Fusarium head blight (FHB), caused by Fusarium graminearum, is a devastating disease of wheat globally. However, the molecular mechanisms underlying the interactions between F. graminearum and wheat remain unclear. Here, we identified a secreted effector protein, FgEC1, that is induced during wheat infection and is required for F. graminearum virulence. FgEC1 suppressed flg22- and chitin-induced callose deposition and reactive oxygen species (ROS) burst in Nicotiana benthamiana. FgEC1 directly interacts with TaGF14b, which is upregulated in wheat heads during F. graminearum infection. Overexpression of TaGF14b increases FHB resistance in wheat without compromising yield. TaGF14b interacts with NADPH oxidase respiratory burst oxidase homolog D (TaRBOHD) and protects it against degradation by the 26S proteasome. FgEC1 inhibited the interaction of TaGF14b with TaRBOHD and promoted TaRBOHD degradation, thereby reducing TaRBOHD-mediated ROS production. Our findings reveal a novel pathogenic mechanism in which a fungal pathogen acts via an effector to reduce TaRBOHD-mediated ROS production.

Analysis of Cytotoxic Granules and Constitutively Produced Extracellular Vesicles from Large Granular Lymphocytic Leukemia Cell Lines.

Large granular lymphocyte leukemias (LGLLs) are rare lymphoproliferative malignancies caused by clonal expansion of granular lymphocytes. T-cell LGLL and natural killer (NK) cell LGLL are defined based on their cellular origin. Their clinical manifestation and pathophysiology vary depending on the subtype and include, e.g., neutropenia, anemia, recurrent infections, and autoimmunity. A limited number of available patient-derived cell lines are considered valuable tools to study the biology of these malignancies. They differ in the expression of lineage-specific surface markers, but generally contain cytotoxic effector molecules in characteristic granules. We investigated the presence and release of lysosome-associated effector proteins in patient-derived LGLL cell lines by flow and imaging cytometry, by Western blotting and by bottom-up proteomics profiling. The tested cell lines did not express FasL (CD178), but did express CD26/DPP4+. Intracellularly, we detected major differences in the abundance and subcellular distribution of granzymes, perforin, and granulysin. Similar differences were seen in enriched lysosome-related effector vesicles (LREVs). The proteomics profiling of enriched EVs from an NK-LGLL line (NKL) and a T-LGLL line (MOTN-1), confirmed individual profiles of effector molecules. Our analyses underscore the individual distribution of effector proteins but also open new routes to define the role of intra- and extracellular granules in the disease manifestation or pathology of LGLLs.

Mechanism of Phyllanthus emblica polyphenols in alleviating DSS-induced ulcerative colitis: Insights from transcriptomics and microbiome analysis

The fruit of Phyllanthus emblica (PE) is a ‘drug homologous food’ that is rich in polyphenols for treating inflammatory diseases. Nevertheless, the ability of PE polyphenols (PEP) to alleviate ulcerative colitis (UC) remains unclear. The aim of this study was to reveal the chemical composition of PEP and its therapeutic potential for treating UC. In this study, PEP was purified with an NKA-2 macroporous resin and identified via liquid chromatography combined with mass spectrometry (LC‒MS). Furthermore, a dextran sulfate sodium salt-induced UC mice model was used to evaluate the ability of PEP to alleviate UC. The symptoms, histopathology, expression of inflammatory cytokines, mucus and tight junction levels, changes in the gut microbiome and colon transcriptomics of UC mice were assessed after PEP intervention. The results suggested that PEP strikingly improved weight loss, disease activity index scores, colon length changes and histopathological damage. Moreover, PEP inhibited the expression of serum proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and sustained intestinal integrity by increasing the expression of Mucin 2, Zonula occludens 1 and Occludin. 16S rRNA sequencing revealed that PEP regulated disorders of the gut microflora related to inflammation and the mucus barrier, including Escherichia_Shigella, Rikenellaceae_RC9_gut_group, Bacteroides, Muribaculaceae, Helicobacter and Anaerostipes. Transcriptome sequencing analysis, western blotting, and immunohistochemistry confirmed that PEP regulated reversed genes as well as effector proteins enriched in the PPAR signaling pathway (PPARγ, MMP1, CYP4A12A, and UCP1) and the Wnt/β-catenin signaling pathway (Wnt8B, FZD9, GSK3β, p-GSK3β, β-catenin, Axin2 and Lgr5). These findings indicate that PEP has therapeutic potential for alleviating UC and could serve as a functional dietary supplement.

Pseudomonas syringae pv. actinidiae Unique Effector HopZ5 Interacts with GF14C to Trigger Plant Immunity.

The bacterial canker of kiwifruit caused by Pseudomonas syringae pv. actinidiae (Psa) is the most devastating disease threatening the global kiwifruit production. This pathogen delivers multiple effector proteins into plant cells to resist plant immune responses and facilitate their survival. Here, we focused on the unique effector HopZ5 in Psa, which previously has been reported to have virulence functions. In this study, our results showed that HopZ5 could cause macroscopic cell death and trigger a serious immune response by agroinfiltration in Nicotiana benthamiana, along with upregulated expression of immunity-related genes and significant accumulation of reactive oxygen species and callose. Subsequently, we confirmed that HopZ5 interacted with the phosphoserine-binding protein GF14C in both the nonhost plant N. benthamiana (NbGF14C) and the host plant kiwifruit (AcGF14C), and silencing of NbGF14C compromised HopZ5-mediated cell death, suggesting that GF14C plays a crucial role in the detection of HopZ5. Further studies showed that overexpression of NbGF14C both markedly reduced the infection of Sclerotinia sclerotiorum and Phytophthora capsica in N. benthamiana, and overexpression of AcGF14C significantly enhanced the resistance of kiwifruit against Psa, indicating that GF14C positively regulates plant immunity. Collectively, our results revealed that the virulence effector HopZ5 could be recognized by plants and interact with GF14C to activate plant immunity.

In silico prediction of effector proteins in oil palm pest, Metisa plana

Metisa plana is one of the main oil palm leaf defoliators in Malaysia. Infestation of M. plana at the oil palm plantation caused severe yield loss in the past. Many control methods have been introduced to combat the infestation, but the problem persists till today. More information regarding the bagworm is needed to understand the pest-host interaction so that an effective control method can be properly designed. This study was carried out to predict the repertoire of effector proteins secreted by M. plana that could potentially suppress the host defence mechanisms. In silico screening of the secretory proteins from predicted genes was done by using several tools namely SignalP, TMHMM, TargetP and WOLFPSORT. Then, the acquired dataset was subjected to functional annotation through a similarity search against UniProt-Trembl database, gene ontology (GO) using BLAST2GO, InterPro protein domain classification, cluster of orthologous groups (COG), and KEGG pathway prediction. To determine the expression of the predicted genes, TPM value was calculated using the available transcriptomic dataset of M. plana. We identified a total of 948 secretory proteins from the initial screening of the 28,738 proteins predicted from BRAKER2 pipeline. Several proteins were classified as specific protein classes associated with effector functions in insect pests, namely detoxifying enzymes, lipases, peroxidases, Ca2+ binding proteins, and chemosensory and odorant binding proteins. This predicted effector data can be used as a reference for future works in validating the protein’s interaction and mechanisms with the oil palm defence responses.

ARGONAUTE2 Localizes to Sites of Sporocysts in the Schistosome-Infected Snail, Biomphalaria glabrata.

MicroRNAs (miRNAs) are a class of small regulatory RNA that are generated via core protein machinery. The miRNAs direct gene-silencing mechanisms to mediate an essential role in gene expression regulation. In mollusks, miRNAs have been demonstrated to be required to regulate gene expression in various biological processes, including normal development, immune responses, reproduction, and stress adaptation. In this study, we aimed to establishment the requirement of the miRNA pathway as part of the molecular response of exposure of Biomphalaria glabrata (snail host) to Schistosoma mansoni (trematode parasite). Initially, the core pieces of miRNA pathway protein machinery, i.e., Drosha, DGCR8, Exportin-5, Ran, and Dicer, together with the central RNA-induced silencing complex (RISC) effector protein Argonaute2 (Ago2) were elucidated from the B. glabrata genome. Following exposure of B. glabrata to S. mansoni miracidia, we identified significant expression up-regulation of all identified pieces of miRNA pathway protein machinery, except for Exportin-5, at 16 h post exposure. For Ago2, we went on to show that the Bgl-Ago2 protein was localized to regions surrounding the sporocysts in the digestive gland of infected snails 20 days post parasite exposure. In addition to documenting elevated miRNA pathway protein machinery expression at the early post-exposure time point, a total of 13 known B. glabrata miRNAs were significantly differentially expressed. Of these thirteen B. glabrata miRNAs responsive to S. mansoni miracidia exposure, five were significantly reduced in their abundance, and correspondingly, these five miRNAs were determined to putatively target six genes with significantly elevated expression and that have been previously associated with immune responses in other animal species, including humans. In conclusion, this study demonstrates the central importance of a functional miRNA pathway in snails, which potentially forms a critical component of the immune response of snails to parasite exposure. Further, the data reported in this study provide additional evidence of the complexity of the molecular response of B. glabrata to S. mansoni infection: a molecular response that could be targeted in the future to overcome parasite infection and, in turn, human schistosomiasis.

Leucine-Rich Repeat Kinases.

Activating mutations in leucine-rich repeat kinase 2 (LRRK2) represent the most common cause of monogenic Parkinson's disease. LRRK2 is a large multidomain protein kinase that phosphorylates a specific subset of the ∼65human Rab GTPases, which are master regulators of the secretory and endocytic pathways. After phosphorylation by LRRK2, Rabs lose the capacity to bind cognate effector proteins and guanine nucleotide exchange factors. Moreover, the phosphorylated Rabs cannot interact with their cognate prenyl-binding retrieval proteins (also known as guanine nucleotide dissociation inhibitors) and, thus, they become trapped on membrane surfaces. Instead, they gain the capacity to bind phospho-Rab-specific effector proteins, such as RILPL1, with resulting pathological consequences. Rab proteins also act upstream of LRRK2 by controlling its activation and recruitment onto membranes. LRRK2 signaling is counteracted by the phosphoprotein phosphatase PPM1H, which selectively dephosphorylates phospho-Rab proteins. We present here our current understanding of the structure, biochemical properties, and cell biology of LRRK2 and its related paralog LRRK1 and discuss how this information guides the generation of LRRK2 inhibitors for the potential benefit of patients.

Abstract Mo139: PDE1, 3 and 4 Inhibition and GPCR Agonism in the Regulation of cAMP Signaling

Modulators of cardiac cyclic adenosine 3’5’-monophosphate (cAMP) and/or its effector protein kinase A (PKA) are used to treat heart disease. Elucidating the mechanisms of endogenous and pharmacological modulators of cAMP, including phosphodiesterase (PDE) inhibitors, is key to predicating alternative drug therapies for heart failure (HF). PDEs regulate confined cAMP/PKA signaling pathways, depending on PDE isoform and localization. PDE3 regulates the SR and sarcolemmal membrane cAMP pools, controlling calcium influx and efflux at these domains. PDE1 regulates sarcolemmal cAMP/PKA signaling. Despite these differences, inhibitors of either increase heart contractility. However, inhibition of PDE3 is pro-arrhythmic, unlike that of PDE1. The regulation of localized cAMP signaling is key to controlled regulation of cell function, with ramifications for HF, and which GPCR lies upstream of PDE1 remains unknown. We hypothesized that the three cAMP-hydrolyzing cardiac PDEs (1, 3, 4) that are found in the human heart would regulate distinct cAMP signaling pathways. Using the cAMP biosensor EPAC-SH187, we performed fluorescence resonance energy transfer experiments in healthy guinea pig adult ventricular myocytes. Cells were treated with inhibitors of PDEs 1, 3, and 4 along with GPCR agonists isoproterenol, glucagon (gcg), and amthamine (amt), respectively for β-adrenergic, gcg and histamine H2 receptors at sub-maximal doses. Cell function and calcium data were obtained using IonOptix. We first asked if PDEi would synergistically regulate pools of cAMP, and found that PDE4i does at pools of cAMP generated by beta-agonism. PDE3i regulates that generated by amt at the H2R, with comparable effects observed in simultaneous or prior H2R stimulation. The combination of these sub-saturating doses exerted strong inotropic and lusitropic effects in paced myocytes. In neither of these domains, PDE1i changed cAMP levels. However, PDE1i intriguingly attenuated the level of gcg-stimulated cAMP production, suggesting a feedback loop mechanism. We conclude that PDEs 1, 3, and 4 regulate distinct pools of cAMP. Importantly, we demonstrate this in a species where the myocardial expression of these PDEs match that of humans as opposed to smaller rodent models.

A novel effector of Aphelenchoides besseyi, AbPFN3, interacts with multiple host proteins to assist parasitic nematode and maintain infection in rice1

The rice white tip nematode (RWTN) Aphelenchoides besseyi secretes effectors that manipulate the cells of its host plant and help the nematode to successfully parasitize and maintain infection in the host. The number of identified RWTN effectors is limited, and the mechanisms of RWTN effectors interacting with plants are largely unknown. Profilins (PFNs) function as hubs that control a complex network of molecular interactions. To gain full knowledge of PFN3 in plant parasitic nematodes, we identified an effector from A. besseyi named AbPFN3. AbPFN3 is transcriptionally upregulated in the juvenile stage of the nematode. In situ hybridization experiments showed that AbPFN3 transcribed in the nematode esophageal glands. Three AbPFN3-interacting proteins (OsAAC1, OsBAP31 and OsSAUR50) were found in the host plant, with interactions occurring in various locations such as the endoplasmic reticulum, cytoplasm, and plasma membrane. Transgenic analyses showed that the expression of AbPFN3 significantly increased plant height and upregulated the expression of AAC1 and BAP31 while downregulating RGA2 and SAUR50. This study describes a new effector protein, AbPFN3, secreted by A. besseyi, that interacts with multiple host proteins. These results suggest the important role of AbPFN3 in host defense response and cell development process.

The processed C-terminus of AvrRps4 effector suppresses plant immunity via targeting multiple WRKYs.

Pathogens generate and secrete effector proteins to the host plant cells during pathogenesis to promote virulence and colonization. If the plant carries resistance (R) proteins that recognize pathogen effectors, effector-triggered immunity (ETI) is activated, resulting in a robust immune response and hypersensitive response (HR). The bipartite effector AvrRps4 from Pseudomonas syringae pv. pisi has been well studied in terms of avirulence function. In planta, AvrRps4 is processed into two parts. The C-terminal fragment of AvrRps4 (AvrRps4C) induces HR in turnip and is recognized by the paired resistance proteins AtRRS1/AtRPS4 in Arabidopsis. Here, we show that AvrRps4C targets a group of Arabidopsis WRKY, including WRKY46, WRKY53, WRKY54, and WRKY70, to induce its virulence function. Indeed, AvrRps4C suppresses the general binding and transcriptional activities of immune-positive regulator WRKY54 and WRKY54-mediated resistance. AvrRps4C interferes with WRKY54's binding activity to target gene SARD1 in vitro, suggesting WRKY54 is sequestered from the SARD1 promoter by AvrRps4C. Through the interaction of AvrRps4C with four WRKYs, AvrRps4 enhances the formation of homo-/heterotypic complexes of four WRKYs and sequesters them in the cytoplasm, thus inhibiting their function in plant immunity. Together, our results provide a detailed virulence mechanism of AvrRps4 through its C-terminus.

A ribonuclease T2 protein FocRnt2 contributes to the virulence of Fusarium oxysporum f. sp. cubense tropical race 4.

Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA invitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.