Spotlight on the Roles of Whitefly Effectors in Insect–Plant Interactions

The Bemisia tabaci species complex (whitefly) causes enormous agricultural losses. These phloem-feeding insects induce feeding damage and transmit a wide range of dangerous plant viruses. Whiteflies colonize a broad range of plant species that appear to be poorly defended against these insects. Substantial research has begun to unravel how phloem feeders modulate plant processes, such as defense pathways, and the central roles of effector proteins, which are deposited into the plant along with the saliva during feeding. Here, we review the current literature on whitefly effectors in light of what is known about the effectors of phloem-feeding insects in general. Further analysis of these effectors may improve our understanding of how these insects establish compatible interactions with plants, whereas the subsequent identification of plant defense processes could lead to improved crop resistance to insects. We focus on the core concepts that define the effectors of phloem-feeding insects, such as the criteria used to identify candidate effectors in sequence-mining pipelines and screens used to analyze the potential roles of these effectors and their targets in planta. We discuss aspects of whitefly effector research that require further exploration, including where effectors localize when injected into plant tissues, whether the effectors target plant processes beyond defense pathways, and the properties of effectors in other insect excretions such as honeydew. Finally, we provide an overview of open issues and how they might be addressed

The impact of diabetes mellitus on survival following resection and adjuvant chemotherapy for pancreatic cancer

BACKGROUND: Diabetes mellitus is frequently observed in pancreatic cancer patients and is both a risk factor and an early manifestation of the disease. METHODS: We analysed the prognostic impact of diabetes on the outcome of pancreatic cancer following resection and adjuvant chemotherapy using individual patient data from three European Study Group for Pancreatic Cancer randomised controlled trials. Analyses were carried out to assess the association between clinical characteristics and the presence of preoperative diabetes, as well as the effect of diabetic status on overall survival. RESULTS: In total, 1105 patients were included in the analysis, of whom 257 (23%) had confirmed diabetes and 848 (77%) did not. Median (95% confidence interval (CI)) unadjusted overall survival in non-diabetic patients was 22.3 (20.8–24.1) months compared with 18.8 (16.9–22.1) months for diabetic patients (P=0.24). Diabetic patients were older, had increased weight and more co-morbidities. Following adjustment, multivariable analysis demonstrated that diabetic patients had an increased risk of death (hazard ratio: 1.19 (95% CI 1.01, 1.40), P=0.034). Maximum tumour size of diabetic patients was larger at randomisation (33.6 vs 29.7 mm, P=0.026). CONCLUSIONS: Diabetes mellitus was associated with increased tumour size and reduced survival following pancreatic cancer resection and adjuvant chemotherapy

The nuclear envelope protein, LAP1B, is a novel protein phosphatase 1 substrate

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.publishe

Transient Expression of Whitefly Effectors in Nicotiana benthamiana Leaves Activates Systemic Immunity Against the Leaf Pathogen Pseudomonas syringae and Soil-Borne Pathogen Ralstonia solanacearum

Infestation of plants with the phloem-feeding whitefly Bemisia tabaci modulates root microbiota and both local and systemic immunity against microbial pathogens. Specifically, aboveground whitefly infestation suppresses pathogen propagation and symptom development caused by the soil-borne pathogens Agrobacterium tumefaciens and Ralstonia solanacearum in the root system through systemic signal transduction. Therefore, we hypothesized that secreted protein(s)/non-protein factors from whitefly saliva (referred to as candidate effectors) might function as insect determinants that activate systemic acquired resistance (SAR) in the host plant. Here, we intensively screened a cDNA library constructed from mRNA from whitefly feeding on Nicotiana benthamiana leaves and selected three candidate effectors 2G4, 2G5, and 6A10, that appear to reduce disease development caused by the aboveground pathogen Pseudomonas syringae pv. tabaci and the soil-borne pathogen R. solanacearum. Transient expression of the three candidate effector cDNAs in leaves primed the expression of SAR marker genes NbPR1a and NbPR2 in local and systemic leaves against P. syringae pv. tabaci, while leaf infiltration with 2G4 or 6A10 cDNA elicited strong defense priming of SAR markers following drench application of R. solanacearum on plant roots. In silico and qRT-PCR analyses revealed the presence of 2G5 and 6A10 transcripts in insect salivary glands. This is the first report of whitefly effectors that prime SAR against aboveground and belowground bacterial pathogens

Data_Sheet_1_Transient Expression of Whitefly Effectors in Nicotiana benthamiana Leaves Activates Systemic Immunity Against the Leaf Pathogen Pseudomonas syringae and Soil-Borne Pathogen Ralstonia solanacearum.PDF

<p>Infestation of plants with the phloem-feeding whitefly Bemisia tabaci modulates root microbiota and both local and systemic immunity against microbial pathogens. Specifically, aboveground whitefly infestation suppresses pathogen propagation and symptom development caused by the soil-borne pathogens Agrobacterium tumefaciens and Ralstonia solanacearum in the root system through systemic signal transduction. Therefore, we hypothesized that secreted protein(s)/non-protein factors from whitefly saliva (referred to as candidate effectors) might function as insect determinants that activate systemic acquired resistance (SAR) in the host plant. Here, we intensively screened a cDNA library constructed from mRNA from whitefly feeding on Nicotiana benthamiana leaves and selected three candidate effectors 2G4, 2G5, and 6A10, that appear to reduce disease development caused by the aboveground pathogen Pseudomonas syringae pv. tabaci and the soil-borne pathogen R. solanacearum. Transient expression of the three candidate effector cDNAs in leaves primed the expression of SAR marker genes NbPR1a and NbPR2 in local and systemic leaves against P. syringae pv. tabaci, while leaf infiltration with 2G4 or 6A10 cDNA elicited strong defense priming of SAR markers following drench application of R. solanacearum on plant roots. In silico and qRT-PCR analyses revealed the presence of 2G5 and 6A10 transcripts in insect salivary glands. This is the first report of whitefly effectors that prime SAR against aboveground and belowground bacterial pathogens.</p

Interaction of Whitefly Effector G4 with Tomato Proteins Impacts Whitefly Performance

The phloem-feeding insect Bemisia tabaci is an important pest, responsible for the transmission of several crop-threatening virus species. While feeding, the insect secretes a cocktail of effectors to modulate plant defense responses. Here, we present a set of proteins identified in an artificial diet on which B. tabaci was salivating. We subsequently studied whether these candidate effectors can play a role in plant immune suppression. Effector G4 was the most robust suppressor of an induced- reactive oxygen species (ROS) response in Nicotiana benthamiana. In addition, G4 was able to suppress ROS production in Solanum lycopersicum (tomato) and Capsicum annuum (pepper). G4 localized predominantly in the endoplasmic reticulum in N. benthamiana leaves and colocalized with two identified target proteins in tomato: REF-like stress related protein 1 (RSP1) and meloidogyne-induced giant cell protein DB141 (MIPDB141). Silencing of MIPDB141 in tomato reduced whitefly fecundity up to 40%, demonstrating that the protein is involved in susceptibility to B. tabaci. Together, our data demonstrate that effector G4 impairs tomato immunity to whiteflies by interfering with ROS production and via an interaction with tomato susceptibility protein MIPDB141. [Graphic: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license

Subcellular distribution of the LAP1B:PP1 complex in HeLa cells.

<p>HeLa cells were transfected with Myc-LAP1B and then processed for immunocytochemistry using specific antibodies to Myc-tag and endogenous lamin B1 and PP1γ and α isoforms. <b>A</b>- Immunolocalization of both myc-LAP1B and lamin B1. <b>B</b>- Immunolocalization of myc-LAP1B and PP1γ and α isoforms. The presence of the complexes is evidenced by the ROI (region of interest). <b>C, D</b>- Confocal profiles representing the green fluorescence intensity (FITC-conjugated secondary antibody labelling Myc-LAP1B) and the red fluorescence intensity (Alexa Fluor 594- conjugated secondary antibody labelling PP1γ [C] or PP1α [D]) in a specific distance (arrow); asterisks denote co-localizing points. <b>E</b>- Quantification of % of co-localization between LAP1B and PP1 isoforms. Values are mean ± SEM, n= 75 cells (for PP1γ) and 55 cells (for PP1α). Photographs were acquired using a LSM 510-Meta confocal microscope. Bars, 10 µm.</p

Co-immunoprecipitation of PP1 binding proteins at the nuclear envelope.

<p>HEK293 cells were immunoprecipitated with PP1 antibody bound to protein A- sepharose beads. The negative controls were performed by incubating cell extracts with beads. IP, immunoprecipitation. IB, immunoblotting.</p

Human LAP1B sequence and domains.

<p><b>A</b>- Nucleotide and corresponding amino acid sequence of human LAP1B encoded by clone 135. Stop and start codons are coloured blue. The three well conserved PP1 binding motifs (RVxF) are highlighted in red (positions 55-59, 212-215 and 538-541 in aa sequence) and a second generic PP1 binding motif (SILK) is coloured green (position 306-309 in aa sequence). The transmembrane domain is highlighted in orange (position 339-361 in aa sequence). <b>B</b>- Schematic illustration of LAP1B domains. Sequence of human LAP1B was aligned against others species using ClustalW algorithm. Sequence conservation is indicated by asterisks (identical sequences), colons (conserved substitutions) and periods (semi-conserved substitutions). BM1, BM2 and BM3, RVxF-like PP1 binding motif 1, 2 and 3, respectively; INM, inner nuclear membrane; ONM, Outer nuclear membrane; SILK, a second generic PP1 binding motif; TM, transmembrane domain.</p

Blot overlay assay with PP1γ1.

<div><p><b>Immunoblotting analysis using a His-tag antibody is also shown</b>. </p> <p>A-Schematic representation of LAP1B deletion mutants cloned into the pET-28c vector. The expected molecular weight (MW) of each construct is indicated. The red boxes represent the RVxF motifs, the green boxes correspond to the SILK motif, and the yellow boxes represent the transmembrane domain. B- Blot overlay assay of full-length LAP1B. Increasing amounts of recombinant full-length LAP1B (12, 24 and 48 µL) were loaded on each well as indicated. C- Blot overlay assay of LAP1B deletion mutants. Deletion mutants: 1, LAP1B–BM1; 2, LAP1B–BM2; 3, LAP1B–BM1/2+TM; 4, LAP1B–BM1/2-TM; 5, LAP1B-BM3+TM; 6, LAP1B-BM3-TM. Non-induced (NI) and pET-28c vector without an insert (pET) were used as negative controls and Nek2A (Nek) as positive control. Bacterial cultures were collected 3 hours after IPTG (1mM) induction at 37°C.</p></div