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. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Discovery of Three Bemisia tabaci Effectors and Their Effect on Gene Expression in Planta.

Bemisia tabaci (whitefly) is a polyphagous agroeconomic pest species complex. Two members of this species complex, Mediterranean (MED) and Middle-East-Asia Minor 1 (MEAM1), have a worldwide distribution and have been shown to manipulate plant defenses through effectors. In this study, we used three different strategies to identify three MEAM1 proteins that can act as effectors. Effector B1 was identified using a bioinformatics-driven effector-mining strategy, whereas effectors S1 and P1 were identified in the saliva of whiteflies collected from artificial diet and in phloem exudate of tomato on which nymphs were feeding, respectively. These three effectors were B. tabaci specific and able to increase whitefly fecundity when transiently expressed in tobacco plants (Nicotiana tabacum). Moreover, they reduced growth of Pseudomonas syringae pv. tabaci in Nicotiana benthamiana. All three effectors changed gene expression in planta, and B1 and S1 also changed phytohormone levels. Gene ontology and KEGG pathway enrichment analysis pinpointed plant-pathogen interaction and photosynthesis as the main enriched pathways for all three effectors. Our data thus show the discovery and validation of three new B. tabaci MEAM1 effectors that increase whitefly fecundity and modulate plant immunity. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Ion Homeostasis and Metabolome Analysis of Arabidopsis 14-3-3 Quadruple Mutants to Salt Stress.

Salinity is one of the major abiotic stresses that limits agricultural productivity worldwide. Many proteins with defined functions in salt stress adaptation are controlled through interactions with members of the 14-3-3 family. In the present study, we generated three 14-3-3 quadruple knockout mutants (qKOs: klpc, klun, and unpc) to study the role of six non-epsilon group 14-3-3 proteins for salt stress adaptation. The relative growth inhibition under 100 mM of NaCl stress was the same for wild-type (Wt) and qKOs, but the accumulation of Na+ in the shoots of klpc was significantly lower than that in Wt. This difference correlated with the higher expression of the HKT1 gene in klpc. Considering the regulatory role of 14-3-3 proteins in metabolism and the effect of salt stress on metabolite accumulation, we analyzed the effect of a 24-h salt treatment on the root metabolome of nutrient solution-grown genotypes. The results indicated that the klpc mutant had metabolome responses that were different from those of Wt. Notably, the reducing sugars, glucose and fructose, were lower in klpc under control and salt stress. On the other hand, their phosphorylated forms, glucose-6P and fructose-6P, were lower under salt stress as compared to Wt. This study provided insight into the functions of the 14-3-3 proteins from non-epsilon group members. In summary, it was found that these proteins control ion homeostasis and metabolite composition under salt stress conditions and non-stressed conditions. The analyses of single, double, and triple mutants that modify subsets from the most effective qKO mutant (klpc) may also reveal the potential redundancy for the observed phenotypes.

Physiological and interactomic analysis reveals versatile functions of Arabidopsis 14-3-3 quadruple mutants in response to Fe deficiency.

To date, few phenotypes have been described for Arabidopsis 14-3-3 mutants or the phenotypes showing the role of 14-3-3 in plant responding to abiotic stress. Although one member of the 14-3-3 protein family (14-3-3 omicron) was shown to be involved in the proper operation of Fe acquisition mechanisms at physiological and gene expression levels in Arabidopsis thaliana, it remains to be explored whether other members play a role in regulating iron acquisition. To more directly and effectively observe whether members of 14-3-3 non-epsilon group have a function in Fe-deficiency adaptation, three higher order quadruple KOs, kappa/lambda/phi/chi (klpc), kappa/lambda/upsilon/nu(klun), and upsilon/nu/phi/chi (unpc) were generated and studied for physiological analysis in this study. The analysis of iron-utilization efficiency, root phenotyping, and transcriptional level of Fe-responsive genes suggested that the mutant with kl background showed different phenotypes from Wt when plants suffered Fe starved, while these phenotypes were absent in the unpc mutant. Moreover, the absence of the four 14-3-3 isoforms in the klun mutant has a clear impact on the 14-3-3 interactome upon Fe deficiency. Dynamics of 14-3-3-client interactions analysis showed that 27 and 17 proteins differentially interacted with 14-3-3 in Wt and klun roots caused by Fe deficiency, respectively. Many of these Fe responsive proteins have a role in glycolysis, oxidative phosphorylation and TCA cycle, the FoF1-synthase and in the cysteine/methionine synthesis. A clear explanation for the observed phenotypes awaits a more detailed analysis of the functional aspects of 14-3-3 binding to the target proteins identified in this study.

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.

Small RNAs from Bemisia tabaci Are Transferred to Solanum lycopersicum Phloem during Feeding.

The phloem-feeding whitefly Bemisia tabaci is a serious pest to a broad range of host plants, including many economically important crops such as tomato. These insects serve as a vector for various devastating plant viruses. It is known that whiteflies are capable of manipulating host-defense responses, potentially mediated by effector molecules in the whitefly saliva. We hypothesized that, beside putative effector proteins, small RNAs (sRNA) are delivered by B. tabaci into the phloem, where they may play a role in manipulating host plant defenses. There is already evidence to suggest that sRNAs can mediate the host-pathogen dialogue. It has been shown that Botrytis cinerea, the causal agent of gray mold disease, takes advantage of the plant sRNA machinery to selectively silence host genes involved in defense signaling. Here we identified sRNAs originating from B. tabaci in the phloem of tomato plants on which they are feeding. sRNAs were isolated and sequenced from tomato phloem of whitefly-infested and control plants as well as from the nymphs themselves, control leaflets, and from the infested leaflets. Using stem-loop RT-PCR, three whitefly sRNAs have been verified to be present in whitefly-infested leaflets that were also present in the whitefly-infested phloem sample. Our results show that whitefly sRNAs are indeed present in tomato tissues upon feeding, and they appear to be mobile in the phloem. Their role in the host-insect interaction can now be investigated.

Light modulated activity of root alkaline/neutral invertase involves the interaction with 14-3-3 proteins.

Alkaline/neutral invertases (A/N-Invs) are now recognized as essential proteins in plant life. They catalyze the irreversible breakdown of sucrose into glucose and fructose and thus supply the cells with energy as well as signaling molecules. In this study we report on a mechanism that affects the activity of the cytosolic invertase AtCINV1 (At-A/N-InvG or AT1G35580). We demonstrate that Ser547 at the extreme C-terminus of the AtCINV1 protein is a substrate of calcium-dependent kinases (CPK3 and 21) and that phosphorylation creates a high-affinity binding site for 14-3-3 proteins. The invertase as such has basal activity, but we provide evidence that interaction with 14-3-3 proteins enhances its activity. The analysis of three quadruple 14-3-3 mutants generated from six T-DNA insertion mutants of the non-epsilon family shows both specificity as well as redundancy for this function of 14-3-3 proteins. The strong reduction in hexose levels in the roots of one 14-3-3 quadruple mutant plant is in line with the activating function of 14-3-3 proteins. The physiological relevance of this mechanism that affects A/N-invertase activity is underscored by the light-induced activation and is another example of the central role of 14-3-3 proteins in mediating dark/light signaling. The nature of the light-induced signal that travels from the shoot to root and the question whether this signal is transmitted via cytosolic Ca(++) changes that activate calcium-dependent kinases, await further study.

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.

Plant 14-3-3 proteins as spiders in a web of phosphorylation.

Protein phosphorylation is essential for many aspects of plant growth and development. To fully modulate the activity of specific proteins after phosphorylation, interaction with members of the 14-3-3 family is necessary. 14-3-3 Proteins are important for many processes because they "assist" a wide range of target proteins with divergent functions. In this review, we will describe how plant 14-3-3 proteins are as spiders in a web of phosphorylation: they act as sensors for phospho-motifs, they themselves are phosphorylated with unknown consequences and they have kinases as target, where some of these phosphorylate 14-3-3 binding motifs in other proteins. Two specific classes of 14-3-3 targets, protein kinases and transcription factors of the bZIP and basic helix-loop-helix-like families, with important and diverse functions in the plant as a whole will be discussed. An important question to be addressed in the near future is how the interaction with 14-3-3 proteins has diverged, both structurally and functionally, between different members of the same protein family, like the kinases and transcription factors.

Protein phosphatase 1γ isoforms linked interactions in the brain.

Posttranslational protein modifications, in particular reversible protein phosphorylation, are important regulatory mechanisms involved in cellular signaling transduction pathways. Thousands of human proteins are phosphorylatable and the tight regulation of phosphorylation states is crucial for cell maintenance and development. Protein phosphorylation occurs primarily on serine, threonine, and tyrosine residues, through the antagonistic actions of protein kinases and phosphatases. The catalytic subunit of protein phosphatase 1 (PP1), a major Ser/Thr-phosphatase, associates with a large variety of regulatory subunits that define substrate specificity and determine specific cellular pathway responses. PP1 has been shown to bind to different proteins in the brain in order to execute key and differential functions. This work reports the identification of proteins expressed in the human brain that interact with PP1γ1 and PP1γ2 isoforms by the yeast two-hybrid method. An extensive search of PP1-binding motifs was performed for the proteins identified, revealing already known PP1 regulators but also novel interactors. Moreover, our results were integrated with the data of PP1γ interacting proteins from several public web databases, permitting the development of physical maps of the novel interactions. The PP1γ interactome thus obtained allowed for the identification of novel PP1 interacting proteins, supporting novel functions of PP1γ isoforms in the human brain.