Two-hybrid Research Articles

Advancements in the Application of Ribosomally Synthesized and Post-Translationally Modified Peptides (RiPPs).

Ribosomally synthesized and post-translationally modified peptides (RiPPs) represent a significant potential for novel therapeutic applications because of their bioactive properties, stability, and specificity. RiPPs are synthesized on ribosomes, followed by intricate post-translational modifications (PTMs), crucial for their diverse structures and functions. PTMs, such as cyclization, methylation, and proteolysis, play crucial roles in enhancing RiPP stability and bioactivity. Advances in synthetic biology and bioinformatics have significantly advanced the field, introducing new methods for RiPP production and engineering. These methods encompass strategies for heterologous expression, genetic refactoring, and exploiting the substrate tolerance of tailoring enzymes to create novel RiPP analogs with improved or entirely new functions. Furthermore, the introduction and implementation of cutting-edge screening methods, including mRNA display, surface display, and two-hybrid systems, have expedited the identification of RiPPs with significant pharmaceutical potential. This comprehensive review not only discusses the current advancements in RiPP research but also the promising opportunities that leveraging these bioactive peptides for therapeutic applications presents, illustrating the synergy between traditional biochemistry and contemporary synthetic biology and genetic engineering approaches.

The BTB-BACK-TAZ domain protein MdBT2 reduces drought resistance by weakening the positive regulatory effect of MdHDZ27 on apple drought tolerance via ubiquitination.

Drought stress is one of the dominating challenges to the growth and productivity in crop plants. Elucidating the molecular mechanisms of plants responses to drought stress is fundamental to improve fruit quality. However, such molecular mechanisms are poorly understood in apple (Malus domestica Borkh.). In this study, we explored that the BTB-BACK-TAZ protein, MdBT2, negatively modulates the drought tolerance of apple plantlets. Moreover, we identified a novel Homeodomain-leucine zipper (HD-Zip) transcription factor, MdHDZ27, using a yeast two-hybrid (Y2H) screen with MdBT2 as the bait. Overexpression of MdHDZ27 in apple plantlets, calli, and tomato plantlets enhanced their drought tolerance by promoting the expression of drought tolerance-related genes [responsive to dehydration 29A (MdRD29A) and MdRD29B]. Biochemical analyses demonstrated that MdHDZ27 directly binds to and activates the promoters of MdRD29A and MdRD29B. Furthermore, in vitro and in vivo assays indicate that MdBT2 interacts with and ubiquitinates MdHDZ27, via the ubiquitin/26S proteasome pathway. This ubiquitination results in the degradation of MdHDZ27 and weakens the transcriptional activation of MdHDZ27 on MdRD29A and MdRD29B. Finally, a series of transgenic analyses in apple plantlets further clarified the role of the relationship between MdBT2 and MdHDZ27, as well as the effect of their interaction on drought resistance in apple plantlets. Collectively, our findings reveal a novel mechanism by which the MdBT2-MdHDZ27 regulatory module controls drought tolerance, which is of great significance for enhancing the drought resistance of apple and other plants.

Generation and characterization of a nanobody against the avian influenza virus H7 subtype

Avian influenza virus (AIV) H7N9 diseases have been recently reported, raising concerns about a potential pandemic. Thus, there is an urgent need for effective therapeutics for AIV H7N9 infections. Herein, camelid immunization and yeast two-hybrid techniques were used to identify potent neutralizing nanobodies (Nbs) targeting the H7 subtype hemagglutinin. First, we evaluated the binding specificity and hemagglutination inhibition activity of the screened Nbs against the H7 subtype hemagglutinin. Nb-Z77, with high hemagglutination inhibition activity was selected from the screened Nbs to optimize the yeast expression conditions and construct oligomeric forms of Nb-Z77 using various ligation methods. The oligomers Nb-Z77-DiGS, Nb-Z77-TriGS, Nb-Z77-Fc and Nb-Z77-Foldon were successfully constructed and expressed. Nb-Z77-DiGS and Nb-Z77-Foldon exhibited considerably greater activity than did Nb-Z77 against H7 subtype hemagglutinin, with median effective concentrations of 384.7 and 27.33 pM and binding affinity values of 213 and 5.21 pM, respectively. Nb-Z77-DiGS and Nb-Z77-Foldon completely inhibited the hemagglutination activity of the inactivated virus H7-Re1 at the lowest concentration of 0.938 μg/mL. This study screened a strain of Nb with high hemagglutination inhibition activity and enhanced its antiviral activity through oligomerization, which may have great potential for developing effective agents for the prevention, diagnosis, and treatment of AIV H7 subtype infection.

Vesicle formation-related protein CaSec16 and its ankyrin protein partner CaANK2B jointly enhance salt tolerance in pepper

Vesicle transport plays important roles in plant tolerance against abiotic stresses. However, the contribution of a vesicle formation related protein CaSec16 (COPII coat assembly protein Sec16-like) in pepper tolerance to salt stress remains unclear. In this study, we report that the expression of CaSec16 was upregulated by salt stress. Compared to the control, the salt tolerance of pepper with CaSec16-silenced was compromised, which was shown by the corresponding phenotypes and physiological indexes, such as the death of growing point, the aggravated leaf wilting, the higher increment of relative electric leakage (REL), the lower content of total chlorophyll, the higher accumulation of dead cells, H2O2, malonaldehyde (MDA), and proline (Pro), and the inhibited induction of marker genes for salt-tolerance and vesicle transport. In contrast, the salt tolerance of pepper was enhanced by the transient overexpression of CaSec16. In addition, heterogeneously induced CaSec16 protein did not enhance the salt tolerance of Escherichia coli, an organism lacking the vesicle transport system. By yeast two-hybrid method, an ankyrin protein, CaANK2B, was identified as the interacting protein of CaSec16. The expression of CaANK2B showed a downward trend during the process of salt stress. Compared with the control, pepper plants with transient-overexpression of CaANK2B displayed increased salt tolerance, whereas those with CaANK2B-silenced exhibited reduced salt tolerance. Taken together, both the vesicle formation related protein CaSec16 and its interaction partner CaANK2B can improve the pepper tolerance to salt stress.

Nesprin-2 is a novel scaffold protein for telethonin and FHL-2 in the cardiomyocyte sarcomere

Nesprins comprise a family of multi-isomeric scaffolding proteins, forming the linker of nucleoskeleton-and-cytoskeleton complex with lamin A/C, emerin and SUN1/2 at the nuclear envelope. Mutations in nesprin-1/-2 are associated with Emery-Dreifuss muscular dystrophy (EDMD) with conduction defects and dilated cardiomyopathy (DCM). We have previously observed sarcomeric staining of nesprin-1/-2 in cardiac and skeletal muscle, but nesprin function in this compartment remains unknown. In this study we show that specific nesprin-2 isoforms are highly expressed in cardiac muscle and localise to the Z-disc and I band of the sarcomere. Expression of GFP-tagged nesprin-2 giant spectrin repeats 52-53, localised to the sarcomere of neonatal rat cardiomyocytes. Yeast two-hybrid screening of a cardiac muscle cDNA library identified telethonin and four and half LIM domain (FHL)-2 as potential nesprin-2 binding partners. GST pull-down and immunoprecipitation confirmed the individual interactions between nesprin-2/telethonin and nesprin-2/FHL-2, and showed that nesprin-2 and telethonin binding was dependent on telethonin phosphorylation status. Importantly, the interactions between these binding partners were impaired by mutations in nesprin-2, telethonin and FHL-2 identified in EDMD with DCM and hypertrophic cardiomyopathy patients. These data suggest that nesprin-2 is a novel sarcomeric scaffold protein that may potentially participate in maintenance and/or regulation of sarcomeric organisation and function.

Characterization of Microbotryum lychnidis-dioicae Secreted Effector Proteins, Their Potential Host Targets, and Localization in a Heterologous Host Plant.

Microbotryum lychnidis-dioicae is an obligate fungal species colonizing the plant host, Silene latifolia. The fungus synthesizes and secretes effector proteins into the plant host during infection to manipulate the host for completion of the fungal lifecycle. The goal of this study was to continue functional characterization of such M. lychnidis-dioicae effectors. Here, we identified three putative effectors and their putative host-plant target proteins. MVLG_02245 is highly upregulated in M. lychnidis-dioicae during infection; yeast two-hybrid analysis suggests it targets a tubulin α-1 chain protein ortholog in the host, Silene latifolia. A potential plant protein interacting with MVLG_06175 was identified as CASP-like protein 2C1 (CASPL2C1), which facilitates the polymerization of the Casparian strip at the endodermal cells. Proteins interacting with MVLG_05122 were identified as CSN5a or 5b, involved in protein turnover. Fluorescently labelled MVLG_06175 and MVLG_05122 were expressed in the heterologous plant, Arabidopsis thaliana. MVLG_06175 formed clustered granules at the tips of trichomes on leaves and in root caps, while MVLG_05122 formed a band structure at the base of leaf trichomes. Plants expressing MVLG_05122 alone were more resistant to infection with Fusarium oxysporum. These results indicate that the fungus might affect the formation of the Casparian strip in the roots and the development of trichomes during infection as well as alter plant innate immunity.

Unraveling Desmin's Head Domain Structure and Function.

Understanding the structure and function of intermediate filaments (IFs) is necessary in order to explain why more than 70 related IF genes have evolved in vertebrates while maintaining such dramatically tissue-specific expression. Desmin is a member of the large multigene family of IF proteins and is specifically expressed in myocytes. In an effort to elucidate its muscle-specific behavior, we have used a yeast two-hybrid system in order to identify desmin's head binding partners. We described a mitochondrial and a lysosomal protein, NADH ubiquinone oxidoreductase core subunit S2 (NDUFS2), and saposin D, respectively, as direct desmin binding partners. In silico analysis indicated that both interactions at the atomic level occur in a very similar way, by the formation of a three-helix bundle with hydrophobic interactions in the interdomain space and hydrogen bonds at R16 and S32 of the desmin head domain. The interactions, confirmed also by GST pull-down assays, indicating the necessity of the desmin head domain and, furthermore, point out its role in function of mitochondria and lysosomes, organelles which are disrupted in myopathies due to desmin head domain mutations.

Unveiling the Biological Function of Phyllostachys edulis FBA6 (PeFBA6) through the Identification of the Fructose-1,6-Bisphosphate Aldolase Gene.

Fructose-1,6-bisphosphate aldolase (FBA) is a pivotal enzyme in various metabolic pathways, including glycolysis, gluconeogenesis, and the Calvin cycle. It plays a critical role in CO2 fixation. Building on previous studies on the FBA gene family in Moso bamboo, our study revealed the biological function of PeFBA6. To identify CSN5 candidate genes, this study conducted a yeast two-hybrid library screening experiment. Subsequently, the interaction between CSN5 and PeFBA6 was verified using yeast two-hybrid and LCI experiments. This investigation uncovered evidence that FBA may undergo deubiquitination to maintain glycolytic stability. To further assess the function of PeFBA6, it was overexpressed in rice. Various parameters were determined, including the light response curve, CO2 response curve, and the levels of glucose, fructose, sucrose, and starch in the leaves of overexpressing rice. The results demonstrated that overexpressed rice exhibited a higher saturation light intensity, net photosynthetic rate, maximum carboxylation rate, respiration rate, and increased levels of glucose, fructose, and starch than wild-type rice. These findings indicated that PeFBA6 not only enhanced the photoprotection ability of rice but also improved the photosynthetic carbon metabolism. Overall, this study enhanced our understanding of the function of FBA and revealed the biological function of PeFBA6, thereby providing a foundation for the development of excellent carbon fixation bamboo varieties through breeding.

Functional Differentiation of the Duplicated Gene BrrCIPK9 in Turnip (Brassica rapa var. rapa).

Gene duplication is a key biological process in the evolutionary history of plants and an important driving force for the diversification of genomic and genetic systems. Interactions between the calcium sensor calcineurin B-like protein (CBL) and its target, CBL-interacting protein kinase (CIPK), play important roles in the plant's response to various environmental stresses. As a food crop with important economic and research value, turnip (Brassica rapa var. rapa) has been well adapted to the environment of the Tibetan Plateau and become a traditional crop in the region. The BrrCIPK9 gene in turnip has not been characterized. In this study, two duplicated genes, BrrCIPK9.1 and BrrCIPK9.2, were screened from the turnip genome. Based on the phylogenetic analysis, BrrCIPK9.1 and BrrCIPK9.2 were found located in different sub-branches on the phylogenetic tree. Real-time fluorescence quantitative PCR analyses revealed their differential expression levels between the leaves and roots and in response to various stress treatments. The differences in their interactions with BrrCBLs were also revealed by yeast two-hybrid analyses. The results indicate that BrrCIPK9.1 and BrrCIPK9.2 have undergone Asparagine-alanine-phenylalanine (NAF) site divergence during turnip evolution, which has resulted in functional differences between them. Furthermore, BrrCIPK9.1 responded to high-pH (pH 8.5) stress, while BrrCIPK9.2 retained its ancestral function (low K+), thus providing further evidence of their functional divergence. These functional divergence genes facilitate turnip's good adaptation to the extreme environment of the Tibetan Plateau. In summary, the results of this study reveal the characteristics of the duplicated BrrCIPK9 genes and provide a basis for further functional studies of BrrCBLs-BrrCIPKs in turnip.

StMAPKK5 Positively Regulates Response to Drought and Salt Stress in Potato.

MAPKKs, as one of the main members of the mitogen-activated protein kinase (MAPK) cascade pathway, are located in the middle of the cascade and are involved in many physiological processes of plant growth and development, as well as stress tolerance. Previous studies have found that StMAPKK5 is responsive to drought and salt stress. To further investigate the function and regulatory mechanism of StMAPKK5 in potato stress response, potato variety 'Atlantic' was subjected to drought and NaCl treatments, and the expression of the StMAPKK5 gene was detected by qRT-PCR. StMAPKK5 overexpression and RNA interference-mediated StMAPKK5 knockdown potato plants were constructed. The relative water content, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities, as well as proline (Pro) and malondialdehyde (MDA) contents of plant leaves, were also assayed under drought and NaCl stress. The StMAPKK5 interacting proteins were identified and validated by yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC). The results showed that the expression of StMAPKK5 was significantly up-regulated under drought and NaCl stress conditions. The StMAPKK5 protein was localized in the nucleus, cytoplasm, and cell membrane. The expression of StMAPKK5 affected the relative water content, the enzymatic activities of SOD, CAT, and POD, and the proline and MDA contents of potatoes under drought and salt stress conditions. These results suggest that StMAPKK5 plays a significant role in regulating drought and salt tolerance in potato crop. Yeast two-hybrid (Y2H) screening identified four interacting proteins: StMYB19, StZFP8, StPUB-like, and StSKIP19. BiFC confirmed the authenticity of the interactions. These findings suggest that StMAPKK5 is crucial for potato growth, development, and response to adversity.

Rice small secreted peptide, OsRALF26, recognized by FERONIA-like receptor 1 induces immunity in rice and Arabidopsis.

Rapid alkalinization factors (RALFs), belonging to a family of small secreted peptides, have been considered as important signaling molecules in diverse biological processes, including immunity. Current studies on RALF-modulated immunity mainly focus on Arabidopsis, but little is reported in crop plants. The rice immune receptor XA21 confers immunity to the bacterial blight pathogen, Xanthomonas oryzae pv. oryzae (Xoo). Here, we pursued functional characterization of rice RALF26 (OsRALF26) up-regulated by Xoo during XA21-mediated immune response. When applied exogenously as a recombinant peptide, OsRALF26 induced a series of immune responses, including pathogenesis-related genes (PRs) induction, reactive oxygen species (ROS) production, and callose deposition in rice and/or Arabidopsis. Transgenic rice and Arabidopsis overexpressing OsRALF26 exhibited significantly enhanced resistance to Xoo and Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), respectively. In yeast two-hybrid, pull-down assays, and co-immunoprecipitation analyses, rice FER-like receptor 1 (OsFLR1) was identified as a receptor of OsRALF26. Transient expression of OsFLR1 in Nicotiana benthamiana leaves displayed significantly increased ROS production and callose deposition after OsRALF26 treatment. Together, we propose that OsRALF26 induced by Xoo in an XA21-dependent manner is perceived by OsFLR1 and may play a novel role in the enforcement of XA21-mediated immunity.

The MYB transcription factor SmMYB113 directly regulates ethylene-dependent flower abscission in eggplant

Flower abscission is an important developmental process that can significantly reduce the yield of horticultural plants. We previously reported that SmMYB113 is a key transcription factor promoting anthocyanin biosynthesis and improve fruit quality. However, the overexpression of SmMYB113 in eggplant increased flower drop rate and reduced fruit yield. Here, we elucidate the regulatory mechanisms of SmMYB113 on flower abscission in eggplant. RNA-seq analysis indicated that the regulation of flower abscission by SmMYB113 was associated with altered expression of genes related to ethylene biosynthesis and signal transduction, including ethylene biosynthetic genes SmACS1, SmACS8 and SmACO4. Then, the ethylene content in flowers and the function of ethephon (ETH, which promotes fruit ripening) and 1-Methylcyclopropene (1-MCP, which acts as an ethylene perception inhibitor) were analyzed, which revealed that SmMYB113 directly regulates ethylene-dependent flower abscission. Yeast one-hybrid and dual-luciferase assays revealed that SmMYB113 could directly bind to the promoters of SmACS1, SmACS8, and SmACO4 to activate their expression. Through construction of a yeast two-hybrid (Y2H) screening library, the protein SmERF38 was found to interact with SmMYB113, and verified by Y2H, bimolecular fluorescence complementation (BiFC), and luciferase complementation assay. Furthermore, dual-luciferase assays showed that SmERF38 enhanced the role of SmMYB113 on the promoters of SmACS1. Our results provided new insight into the molecular mechanism of flower abscission in eggplant.

PagARGOS promotes low-lignin wood formation in poplar.

Wood formation, which occurs mainly through secondary xylem development, is important not only for supplying raw material for the 'ligno-chemical' industry but also for driving the storage of carbon. However, the complex mechanisms underlying the promotion of xylem formation remain to be elucidated. Here, we found that overexpression of Auxin-Regulated Gene involved in Organ Size (ARGOS) in hybrid poplar 84 K (Populus alba × Populus tremula var. glandulosa) enlarged organ size. In particular, PagARGOS promoted secondary growth of stems with increased xylem formation. To gain further insight into how PagARGOS regulates xylem development, we further carried out yeast two-hybrid screening and identified that the auxin transporter WALLS ARE THIN1 (WAT1) interacts with PagARGOS. Overexpression of PagARGOS up-regulated WAT1, activating a downstream auxin response promoting cambial cell division and xylem differentiation for wood formation. Moreover, overexpressing PagARGOS caused not only higher wood yield but also lower lignin content compared with wild-type controls. PagARGOS is therefore a potential candidate gene for engineering fast-growing and low-lignin trees with improved biomass production.

Overexpression of NtGPX8a Improved Cadmium Accumulation and Tolerance in Tobacco (Nicotiana tabacum L.).

Cadmium (Cd)-induced oxidative stress detrimentally affects hyperaccumulator growth, thereby diminishing the efficacy of phytoremediation technology aimed at Cd pollution abatement. In the domain of plant antioxidant mechanisms, the role of glutathione peroxidase (GPX) in conferring Cd tolerance to tobacco (Nicotiana tabacum) remained unclear. Our investigation employed genome-wide analysis to identify 14 NtGPX genes in tobacco, revealing their organization into seven subgroups characterized by analogous conserved domain patterns. Notably, qPCR analysis highlighted NtGPX8a as markedly responsive to Cd2+ stress. Subsequent exploration through yeast two-hybridization unveiled NtGPX8a's utilization of thioredoxins AtTrxZ and AtTrxm2 as electron donors, and without interaction with AtTrx5. Introduction of NtGPX8a into Escherichia coli significantly ameliorated Cd-induced adverse effects on bacterial growth. Transgenic tobacco overexpressing NtGPX8a demonstrated significantly augmented activities of GPX, SOD, POD, and CAT under Cd2+ stress compared to the wild type (WT). Conversely, these transgenic plants exhibited markedly reduced levels of MDA, H2O2, and proline. Intriguingly, the expression of NtGPX8a in both E. coli and transgenic tobacco led to increased Cd accumulation, confirming its dual role in enhancing Cd tolerance and accumulation. Consequently, NtGPX8a emerges as a promising candidate gene for engineering transgenic hyperaccumulators endowed with robust tolerance for Cd-contaminated phytoremediation.

Chigno/CG11180 and SUMO are Chinmo-interacting proteins with a role in Drosophila testes somatic support cells.

Stem cells are critical for replenishment of cells lost to death, damage or differentiation. Drosophila testes are a key model system for elucidating mechanisms regulating stem cell maintenance and differentiation. An intriguing gene identified through such studies is the transcription factor, chronologically inappropriate morphogenesis (Chinmo). Chinmo is a downstream effector of the Jak-STAT signaling pathway that acts in testis somatic stem cells to ensure maintenance of male stem cell fate and sexual identity. Defects in these processes can lead to infertility and the formation of germ cell tumors. While Chinmo's effect on testis stem cell behavior has been investigated in detail, there is still much to be learned about its structure, function, and interactions with other proteins. Using a two-hybrid screen, we find that Chinmo interacts with itself, the small ubiquitin-like modifier SUMO, the novel protein CG11180, and four other proteins (CG4318, Ova (ovaries absent), Taf3 (TBP-associated factor 3), and CG18269). Since both Chinmo and CG11180 contain sumoylation sites and SUMO-interacting motifs (SIMs), we analyzed their interaction in more detail. Using site-directed mutagenesis of a unique SIM in CG11180, we demonstrate that Chinmo's interaction with CG11180 is SUMO-dependent. Furthermore, to assess the functional relevance of both SUMO and CG11180, we performed RNAi-mediated knockdown of both proteins in somatic cells of the Drosophila testis. Using this approach, we find that CG11180 and SUMO are required in somatic cells of adult testes, and that reduction of either protein causes formation of germ cell tumors. Overall, our work suggests that SUMO may be involved in the interaction of Chinmo and CG11180 and that these genes are required in somatic cells of the adult Drosophila testis. Consistent with the CG11180 knockdown phenotype in male testes, and to underscore its connection to Chinmo, we propose the name Chigno (Childless Gambino) for CG11180.

The RING zinc finger protein LbRZF1 promotes salt gland development and salt tolerance in Limonium bicolor.

The recretohalophyte Limonium bicolor thrives in high-salinity environments because salt glands on the above-ground parts of the plant help to expel excess salt. Here, we characterize a nucleus-localized C3HC4 (RING-HC)-type zinc finger protein of L. bicolor named RING ZINC FINGERPROTEIN 1 (LbRZF1). LbRZF1 was expressed in salt glands and in response to NaCl treatment. LbRZF1 showed no E3 ubiquitin ligase activity. The phenotypes of overexpression and knockout lines for LbRZF1 indicated that LbRZF1 positively regulated salt gland development and salt tolerance in L. bicolor. lbrzf1 mutants had fewer salt glands and secreted less salt than did the wild-type, whereas LbRZF1-overexpressing lines had opposite phenotypes, in keeping with the overall salt tolerance of these plants. A yeast two-hybrid screen revealed that LbRZF1 interacted with LbCATALASE2 (LbCAT2) and the transcription factor LbMYB113, leading to their stabilization. Silencing of LbCAT2 or LbMYB113 decreased salt gland density and salt tolerance. The heterologous expression of LbRZF1 in Arabidopsis thaliana conferred salt tolerance to this non-halophyte. We also identified the transcription factor LbMYB48 as an upstream regulator of LbRZF1 transcription. The study of LbRZF1 in the regulation network of salt gland development also provides a good foundation for transforming crops and improving their salt resistance.

A selection and optimization strategy for single-domain antibodies targeting the PHF6 linear peptide within the tau intrinsically disordered protein

The use of VHHs (Variable domain of the Heavy-chain of the Heavy-chain-only antibodies) as disease-modifying biomolecules in neurodegenerative disorders holds promises including targeting of aggregation-sensitive proteins. Exploitation of their clinical values depends however on the capacity to deliver VHHs with optimal physico-chemical properties for their specific context of use. We described previously a VHH with high therapeutic potential in a family of neurodegenerative diseases called tauopathies. The activity of this promising parent VHH named Z70 relies on its binding within the central region of the Tau protein. Accordingly, we carried out random mutagenesis followed by yeast two-hybrid screening to obtain optimized variants. The VHHs selected from this initial screen targeted the same epitope as VHH Z70 as shown using nuclear magnetic resonance spectroscopy and had indeed improved binding affinities according to dissociation constant values obtained by surface plasmon resonance spectroscopy. The improved affinities can be partially rationalized based on three-dimensional structures and NMR data of three complexes consisting of an optimized VHH and a peptide containing the Tau epitope. Interestingly, the ability of the VHH variants to inhibit Tau aggregation and seeding could not be predicted from their affinity alone. We indeed showed that the in vitro and in cellulo VHH stabilities are other limiting key factors to their efficacy. Our results demonstrate that only a complete pipeline of experiments, here described, permits a rational selection of optimized VHH variants, resulting in the selection of VHH variants with higher affinities and/or acting against Tau seeding in cell models.

PEX1 is essential for glycosome biogenesis and trypanosomatid parasite survival.

Trypanosomatid parasites are kinetoplastid protists that compartmentalize glycolytic enzymes in unique peroxisome-related organelles called glycosomes. The heterohexameric AAA-ATPase complex of PEX1-PEX6 is anchored to the peroxisomal membrane and functions in the export of matrix protein import receptor PEX5 from the peroxisomal membrane. Defects in PEX1, PEX6 or their membrane anchor causes dysfunction of peroxisomal matrix protein import cycle. In this study, we functionally characterized a putative Trypanosoma PEX1 orthologue by bioinformatic and experimental approaches and show that it is a true PEX1 orthologue. Using yeast two-hybrid analysis, we demonstrate that TbPEX1 can bind to TbPEX6. Endogenously tagged TbPEX1 localizes to glycosomes in the T. brucei parasites. Depletion of PEX1 gene expression by RNA interference causes lethality to the bloodstream form trypanosomes, due to a partial mislocalization of glycosomal enzymes to the cytosol and ATP depletion. TbPEX1 RNAi leads to a selective proteasomal degradation of both matrix protein import receptors TbPEX5 and TbPEX7. Unlike in yeast, PEX1 depletion did not result in an accumulation of ubiquitinated TbPEX5 in trypanosomes. As PEX1 turned out to be essential for trypanosomatid parasites, it could provide a suitable drug target for parasitic diseases. The results also suggest that these parasites possess a highly efficient quality control mechanism that exports the import receptors from glycosomes to the cytosol in the absence of a functional TbPEX1-TbPEX6 complex.

The glycoside hydrolase 7 member VdGH7a regulates Verticillium dahliae pathogenicity and induces host defenses by interacting with GhOLP11

Pathogens secrete multiple enzymes that can degrade the cell wall, thereby weakening the host’s cell wall and facilitating the penetration of the pathogen into the plant. In this study, we identified VdGH7a, a glycoside hydrolase family 7 (GH7) cellobiohydrolase from Verticillium dahliae, which exhibited hydrolytic activity against 1,4-β-glucan. Interestingly, we found that VdGH7a induced cell death in Nicotiana benthamiana when signal peptides were present. However, this phenomenon was effectively prevented by the carbohydrate-binding type-1 (CBM1) protein domain. Furthermore, we observed that the knockout of VdGH7a significantly reduced the pathogenicity of V. dahliae to cotton plant, as evidenced by the inability of the knockout mutants to penetrate cellophane membrane. Additionally, these knockout mutants displayed diminished ability to exploit carbon sources, rendering them more susceptible to osmotic and cell wall stresses. Moreover, VdGH7a interacted with an osmotin-like protein (GhOLP1) in cotton through yeast two-hybrid screening, and further confirmed using bi-molecular fluorescence complementation (BiFC) and luciferase complementation imaging (LCI). Furthermore, virus-induced gene silencing technology was employed to silence GhOLP1, causing cotton’s salicylic acid (SA) content and resistance to V. dahliae were both reduced, whereas heterologous overexpression of GhOLP1 in Arabidopsis increased both resistance and the expression of genes involved in the SA signaling pathway. Collectively, these findings demonstrate a virulence strategy whereby the secreted protein VdGH7a from V. dahliae interacts with GhOLP1 to stimulate host immunity and play a significant role in plant resistance against V. dahliae.

MoAti1 mediates mitophagy by facilitating recruitment of MoAtg8 to promote invasive growth in Magnaporthe oryzae.

Mitophagy is a selective autophagy for the degradation of damaged or excessive mitochondria to maintain intracellular homeostasis. In Magnaporthe oryzae, a filamentous ascomycetous fungus that causes rice blast, the most devastating disease of rice, mitophagy occurs in the invasive hyphae to promote infection. To date, only a few proteins are known to participate in mitophagy and the mechanisms of mitophagy are largely unknown in pathogenic fungi. Here, by a yeast two-hybrid screen with the core autophagy-related protein MoAtg8 as a bait, we obtained a MoAtg8 interactor MoAti1 (MoAtg8-interacting protein 1). Fluorescent observations and protease digestion analyses revealed that MoAti1 is primarily localized to the peripheral mitochondrialouter membrane and is responsible for recruiting MoAtg8 to mitochondria under mitophagy induction conditions. MoAti1 is specifically required for mitophagy, but not for macroautophagy and pexophagy. Infection assays suggested that MoAti1 is required for mitophagy in invasive hyphae during pathogenesis. Notably, no homologues of MoAti1 were found in rice and human protein databases, indicating that MoAti1 may be used as a potential target to control rice blast. By the host-induced gene silencing (HIGS) strategy, transgenic rice plants targeted to silencing MoATI1 showed enhanced resistance against M. oryzae with unchanged agronomic traits. Our results suggest that MoATI1 is required for mitophagy and pathogenicity in M. oryzae and can be used as a target for reducing rice blast.