Small Protein Research Articles

Genome-wide identification and characterisation of hevein precursor genes in Hevea brasiliensis

Hevea brasiliensis is the primary source of natural rubber, which is obtained from its latex and used in the manufacturing of various products. Hevein is a small protein found in the latex, produced by post-translational cleavage of prohevin, which is encoded by the hevein precursor gene. It exhibits antimicrobial and agglutination properties. Several nucleotide sequences that encode the hevein precursor genes were reported, however, the sequences are not characterised well. In the present study, four full-length hevein precursor sequences (Hevein 1–4) were obtained by manually curating the sequences from different databases. All sequences show high homology with the highest identity between Hevein 1 and Hevein 3. In a phylogenetic analysis along with sequences from related plants, all sequences from H. brasiliensis were clustered into a specific clade. All hevein precursor genes were expressed in the latex samples obtained from three clones; RRIM 600, RRIM 3001 and PB 350 of less than one year old and five-year-old RRIM 3001 plants, grown in plant house, as well as field-grown trees of clone PB 350 belonging to three different ages; two years, six years and fifteen years. All hevein precursor genes in the five-year-old RRIM 3001 plants with stunted growth, maintained in the plant house showed alternative splicing. Hevein 3 was expressed with two splice variants, one with intron retention and the other without intron whereas the other genes were expressed with only intron retained variant. Differential expression analysis using nanoplate digital PCR showed that Hevein 2 and Hevein 3 were expressed with no significant difference among the three young H. brasiliensis clones. The expression of Hevein 2 and Hevein 3 among the H. brasiliensis clone PB 350 of different ages grown in field conditions showed significant difference. The present study provides a better understanding on the importance of hevein precursor genes in different physiological responses which will be useful for further research leading to the genetic improvement of H. brasiliensis.

Small proteins, great promises: Geographic bioprospecting of Bowman–Birk protease inhibitors and domestication side‐effects in African cowpea (Vigna unguiculata L.)

Societal Impact StatementThe legume crop cowpea is grown worldwide, but 90% of the world's total share is produced in Africa. It is a promising species due to its resilience properties, balance of macro and micronutrients and presence of health‐promoting bioactive compounds. In African countries, cowpea has a crucial role in guaranteeing food security as a subsistence crop for families and commercial income for small farmers. The discovery of compounds with high nutraceutical value and bioactive properties supports socio‐economic policies to improve health and nutrition, especially in low‐ and middle‐income countries. In turn, this encourages biodiversity protection and crop enhancement programmes.Summary Bowman–Birk protease inhibitors (BBIs) are a restricted group of small proteins in plants mainly involved in defence mechanisms against pests. BBIs are demonstrated to be active components capable of reducing the viabilities of different cancer cell lines. BBI bioactivity is directly linked to the inhibition capacity, but the variability and the efficiency against the physiological targets of different BBI isoforms remain still unexplored. We analysed the natural genetic diversity of two main genes encoding BBI trypsin‐trypsin (BBI‐TT) and trypsin‐chymotrypsin (BBI‐TC) in wild and domesticated cowpea samples mainly spread in Sub‐Saharan Africa. We analysed DNA sequences and respective amino acidic isoforms/isoproteins to explore signs of natural selection and haplotype relationships. Moreover, we calculated the binding energy between BBIs and their biological targets to identify which are the most efficient inhibitors and their geographical locations. We found a high level of haplotype diversity for both genes, almost exclusively in wild accessions and detected positive and negative selection signals in the amino acid sequences. Furthermore, in the wild diversity pool, some BBI‐TT and BBI‐TC mature proteins were potentially better interactors with the physiological targets. The long interaction between plant‐pathogen has selected new and useful isoforms in wild lineages that have allowed the chances of survival of the species to improve. On the other hand, the domestication process has produced an intense bottleneck leaving only poorly efficient BBI variants. In addition to providing information on the natural diversity and evolution of BBIs, our work discusses the potential applications in agriculture and human health.

Computational insight into the spectroscopic and molecular docking analysis of estrogen receptor with ligand 2,3-dimethyl-N[2-(hydroxy)benzylidene] aniline

A comprehensive approach encompassing a blend of spectral techniques and Density Functional Theory (DFT) was employed to elucidate the electronic, vibrational, structural, and nonlinear optical properties of 2,3-dimethyl-N[2-(Hydroxy)benzylidene] aniline (DNHBA). The investigation of this chemical compound involved a meticulous examination using spectroscopic techniques (FTIR, FT-Raman). The DFT calculations utilized the B3LYP method in conjunction with 6-311G (d, p) basis sets. The findings were substantiated by comparing the estimated vibrational frequencies and molecule geometry with experimental data. Using the Time Dependent −Density Functional Theory (TD-DFT) method to calculate the energy band gap of the compound at different solvents. We looked into frontier molecular orbitals (FMO) Molecular electrostatic potential (MEP) and natural bond analysis (NBO) to determine the compound’s kinetic stability and chemical reactivity. Hirshfeld surface (HS) analysis calculated the O–H..O, H-H and intermolecular interaction energy of the compound. The electron density distribution, interactions, and excitation within the title molecule are illustrated by performing topological analyses (RDG, ELF and LOL) using the Multiwfn software. In addition to these assessments, molecular dynamics provides an understanding of the system's dynamic development over a predefined duration during which atoms and molecules are permitted to interact. Utilizing the molecular docking approach allows us to energize the atomic-level interplay between small molecules and proteins within the binding sites of target proteins. This approach enhances our comprehension of vital biochemical processes associated with the behavior of small molecule. The intricacy and hazards associated with drug discovery and development processes have increased dramatically, leading to higher drug research costs.

Design and Biological Evaluation of the Long-Acting C5-Inhibited Ornithodoros moubata Complement Inhibitor (OmCI) Modified with Fatty Acid.

Disorder of complement response is a significant pathogenic factor causing some autoimmune and inflammation diseases. The Ornithodoros moubata Complement Inhibitor (OmCI), a small 17 kDa natural protein, was initially extracted from soft tick salivary glands. The protein was found binding to complement C5 specifically, inhibiting the activation of the complement pathway, which is a successful therapeutic basis of complement-mediated diseases. However, a short half-life due to rapid renal clearance is a common limitation of small proteins for clinical application. In this study, we extended the half-life of OmCI by modifying it with fatty acid, which was a method used to improve the pharmacokinetics of native peptides and proteins. Five OmCI mutants were initially designed, and single-site cysteine mutation was introduced to each of them. After purification, four OmCI mutants were obtained that showed similar in vitro biological activities. Three mutants of them were subsequently coupled with different fatty acids by nucleophilic substitution. In total, 15 modified derivatives were screened and tested for anticomplement activity in vitro. The results showed that coupling with fatty acid would not significantly affect their complement-inhibitory activity (CH50 and AH50). OmCIT90C-CM02 and OmCIT90C-CM05 were validated as the applicable OmCI bioconjugates for further pharmacokinetic assessments, and both showed improved plasma half-life in mice compared with unmodified OmCI (15.86, 17.96 vs 2.57 h). In summary, our data demonstrated that OmCI conjugated with fatty acid could be developed as the potential long-acting C5 complement inhibitor in the clinic.

Genetic characterization, structural analysis, and detection of positive selection in small heat shock proteins of Cypriniformes and Clupeiformes.

In the current investigation, a total of 42 full-length, non-redundant small heat shock proteins (sHsp) were detected in Cyprinus carpio, Labeo rohita, Danio rerio, Salmo salar, Oncorhynchus mykiss, and Clupea harengus. The sHsp genes were classified into three groups based on phylogenetic analysis. All the sHsps were shown to have higher aliphatic index values, which is an indication that these proteins are more thermally stable. The hydrophilic nature of sHsps was deduced from the fact that all fish species had negative GRAVY scores. In all of the representative fish species, sHsp genes were assigned to distinct chromosomes in an inconsistent and unequal manner. Segmental duplications are the main events that have contributed to the expansion of the sHsp genes in all species. We were also able to determine the selective pressure that was placed on particular codons and discovered several significant coding sites within the coding region of sHsps. Eventually, diversifying positive selection was found to be connected with evolutionary changes in sHsp proteins, which showed that gene evolution controlled the fish adaption event in response to environmental conditions. Clarification of the links between sHsps and environmental stress in fish will be achieved through rigorous genomic comparison, which will also yield substantial new insights.

Proteomic Identification of Small Extracellular Vesicle Proteins LAMB1 and Histone H4 for Prostate Cancer Diagnosis and Risk Stratification.

Diagnosis and stratification of prostate cancer (PCa) patients using the prostate-specific antigen (PSA) test is challenging. Extracellular vesicles (EVs), as a new star of liquid biopsy, has attracted interest to complement inaccurate PSA screening and invasiveness of tissue biopsy. In this study, a panel of potential small EV (sEV) protein biomarkers is identified from PCa cell lines using label-free LC-MS/MS proteomics. These biomarkers underwent further validation with plasma and urine samples from different PCa stages through parallel reaction monitoring-based targeted proteomics, western blotting, and ELISA. Additionally, a tissue microarray containing cancerous and noncancerous tissues is screened to provide additional evidence of selected sEV proteins associated with cancer origin. Results indicate that sEV protein LAMB1 is highly expressed in human plasma of metastatic PCa patients compared with localised PCa patients and control subjects, while sEV protein Histone H4 is highly expressed in human urine of high-risk PCa patients compared to low-risk PCa patients and control subjects. These two sEV proteins demonstrate higher specificity and sensitivity than the PSA test and show promise for metastatic PCa diagnosis, progression monitoring, and risk stratification.

The clade III subfamily of OsSWEETs directly suppresses rice immunity by interacting with OsHMGB1 and OsHsp20L.

The clade III subfamily of OsSWEETs includes transmembrane proteins necessary for susceptibility to bacterial blight (BB). These genes are targeted by the specific transcription activator-like effector (TALE) of Xanthomonas oryzae pv. oryzae and mediate sucrose efflux for bacterial proliferation. However, the mechanism through which OsSWEETs regulate rice immunity has not been fully elucidated. Here, we demonstrated that the cytosolic carboxyl terminus of OsSWEET11a/Xa13 is required for complementing susceptibility to PXO99 in IRBB13 (xa13/xa13). Interestingly, the C-terminus of ZmXa13, the maize homologue of OsSWEET11a/Xa13, could perfectly substitute for the C-terminus of OsSWEET11a/Xa13. Furthermore, OsSWEET11a/Xa13 interacted with the high-mobility group B1 (OsHMGB1) protein and the small heat shock-like protein OsHsp20L through the same regions in the C-terminus. Consistent with the physical interactions, knockdown or knockout of either OsHMGB1 or OsHsp20L caused an enhanced PXO99-resistant phenotype similar to that of OsSWEET11a/OsXa13. Surprisingly, the plants in which OsHMGB1 or OsHsp20L was repressed developed increased resistance to PXO86, PXO61 and YN24, which carry TALEs targeting OsSWEET14/Xa41 or OsSWEET11a/Xa13. Additionally, OsHsp20L can interact with all six members of clade III OsSWEETs, whereas OsHMGB1 can interact with five other members in addition to OsSWEET12. Overall, we revealed that OsHMGB1 and OsHsp20L mediate conserved BB susceptibility by interacting with clade III OsSWEETs, which are candidates for breeding broad-spectrum disease-resistant rice.

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

Abstract SY12-01: Targeting validated but un-drugged oncogenes with small molecule protein degraders

De novo design of drug-binding proteins with predictable binding energy and specificity.

The de novo design of small molecule-binding proteins has seen exciting recent progress; however, high-affinity binding and tunable specificity typically require laborious screening and optimization after computational design. We developed a computational procedure to design a protein that recognizes a common pharmacophore in a series of poly(ADP-ribose) polymerase-1 inhibitors. One of three designed proteins bound different inhibitors with affinities ranging from <5 nM to low micromolar. X-ray crystal structures confirmed the accuracy of the designed protein-drug interactions. Molecular dynamics simulations informed the role of water in binding. Binding free energy calculations performed directly on the designed models were in excellent agreement with the experimentally measured affinities. We conclude that de novo design of high-affinity small molecule-binding proteins with tuned interaction energies is feasible entirely from computation.

In-house Extraction and Purification of Pfu-Sso7d, a High-processivity DNA Polymerase.

The polymerase chain reaction (PCR) is an extensively used technique to quickly and accurately make many copies of a specific segment of DNA. In addition to naturally existing DNA polymerases, PCR utilizes a range of genetically modified recombinant DNA polymerases, each characterized by varying levels of processivity and fidelity. Pfu-Sso7d, a fusion DNA polymerase, is obtained by the fusion of Sso7d, a small DNA-binding protein, with Pfu DNA polymerase. Pfu-Sso7d is known for its high processivity, efficiency, and fidelity but is sold at a sumptuously high price under various trade names and commercial variants. We recently reported a quick and easy purification protocol that utilizes ethanol or acetone to precipitate Pfu-Sso7d from heat-cleared lysates. We also optimized a PCR buffer solution that outperforms commercial buffers when used with Pfu-Sso7d. Here, we provide a step-by-step guide on how to purify recombinant Pfu-Sso7d. This purification protocol and the buffer system will offer researchers cost-efficient access to fusion polymerase. Key features • We detail a precipitation-based protocol utilizing ethanol and acetone for purifying Pfu-Sso7d. • Despite ethanol and acetone displaying effective precipitation efficiency, acetone is preferred for its superior performance. • Furthermore, we present a PCR buffer that outperforms commercially available PCR buffers. • The Pfu-Sso7d purified in-house and the described PCR buffer exhibit excellent performance in PCRapplications.

Abstract LB045: Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates

Abstract The placental alkaline phosphatases ALPP and ALPPL2 are attractive targets for antibody and protein-drug conjugate (ADC and PDC) approaches. These GPI-anchored cell-surface proteins, which share very high sequence identity, are over-expressed in a number of different solid tumor indications including testicular, ovarian, endometrial, gastric, pancreatic and non-small cell lung cancers but have very little expression on normal adult tissues. Consequently, there is significant interest in exploiting ALPP/ALPPL2 for the development of new targeted oncology therapeutics, and an ALPP/ALPPL2 ADC, utilizing a classical full-length IgG antibody, is currently in Phase I clinical trials. The exploitation of biparatopic binding is an attractive approach for improving the therapeutic index of ADCs. This mode of target engagement increases the effective number of binding sites on the tumor cells and can promote internalisation and trafficking, thereby delivering increased efficacy for the same payload bolus and opening up patient populations with lower target expression. This can lead to a widening of the therapeutic window, by improving the efficacy achievable for a given amount of payload administered (often defined as the “platform” toxicity). For the development of biparatopic conjugates, the use of small protein domain binders offers advantages over the use of full-length antibodies, as they can readily be reformatted into a variety of biparatopic architectures to deliver homogeneous drug conjugates in high yield. Additionally, smaller protein formats have the capacity to penetrate deeper into solid tumors than large antibodies. We have identified a series of highly selective, high affinity ALPP/ALPPL2 single domain VHH binders, which target distinct epitopes on the protein and which, importantly, show no binding to the closely related ALPI or ALPL isoforms (which have high normal tissue expression). Using protein engineering approaches, a series of homogenous site-specifically labelled biparatopic PDCs have been developed employing the clinically validated vcMMAE (vedotin) payload. In pre-clinical studies, these PDC agents demonstrate excellent anti-tumor efficacy in vivo in ALPP/ALPPL2 positive cell-line derived xenograft models of gastric, pancreatic and ovarian cancer displaying a range of target expression. In benchmarking studies, lead biparatopic PDCs show increased internalisation and target-mediated in vitro cell kill potency compared to an ALPP/ALPPL2 targeting ADC. Additionally, in multiple CDX models, lead biparatopic PDCs demonstrate improved efficacy over an ALPP/ALPPL2 ADC both on a mg/kg and on a payload equivalents basis, in both single dose and multi-dose studies, providing complete regressions. Based on this excellent efficacy profile and favourable developability characteristics, preclinical development activities have been initiated. Citation Format: Graham Cotton, Estelle G. McLean, Paul Trumper, Mark Wappett, Stephanie Gatdula, Stacey Bell, Greg Papadakos, Kieron Lucas, Stephanie Burton, Chiara Saladino, Georgiana Parau, Aidan McCann, Jennifer Thom, Aaron N. Cranston, Tim Harrison. Exploiting biparatopic binding to deliver novel, differentiated ALPP/ALPPL2 targeting protein drug conjugates [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB045.

Improving the pharmacokinetics, biodistribution and plasma stability of monobodies.

Cancer is a leading cause of death worldwide. Several targeted anticancer drugs entered clinical practice and improved survival of cancer patients with selected tumor types, but therapy resistance and metastatic disease remains a challenge. A major class of targeted anticancer drugs are therapeutic antibodies, but their use is limited to extracellular targets. Hence, alternative binding scaffolds have been investigated for intracellular use and better tumor tissue penetration. Among those, monobodies are small synthetic protein binders that were engineered to bind with high affinity and selectivity to central intracellular oncoproteins and inhibit their signaling. Despite their use as basic research tools, the potential of monobodies as protein therapeutics remains to be explored. In particular, the pharmacological properties of monobodies, including plasma stability, toxicity and pharmacokinetics have not been investigated. Here, we show that monobodies have high plasma stability, are well-tolerated in mice, but have a short half-life in vivo due to rapid renal clearance. Therefore, we engineered monobody fusions with an albumin-binding domain (ABD), which showed enhanced pharmacological properties without affecting their target binding: We found that ABD-monobody fusions display increased stability in mouse plasma. Most importantly, ABD-monobodies have a dramatically prolonged in vivo half-life and are not rapidly excreted by renal clearance, remaining in the blood significantly longer, while not accumulating in specific internal organs. Our results demonstrate the promise and versatility of monobodies to be developed into future therapeutics for cancer treatment. We anticipate that monobodies may be able to extend the spectrum of intracellular targets, resulting in a significant benefit to patient outcome.

Enhanced therapeutic potential of antibody fragment via IEDDA-mediated site-specific albumin conjugation

BackgroundThe use of single-chain variable fragments (scFvs) for treating human diseases, such as cancer and immune system disorders, has attracted significant attention. However, a critical drawback of scFv is its extremely short serum half-life, which limits its therapeutic potential. Thus, there is a critical need to prolong the serum half-life of the scFv for clinical applications. One promising serum half-life extender for therapeutic proteins is human serum albumin (HSA), which is the most abundant protein in human serum, known to have an exceptionally long serum half-life. However, conjugating a macromolecular half-life extender to a small protein, such as scFv, often results in a significant loss of its critical properties.ResultsIn this study, we conjugated the HSA to a permissive site of scFv to improve pharmacokinetic profiles. To ensure minimal damage to the antigen-binding capacity of scFv upon HSA conjugation, we employed a site-specific conjugation approach using a heterobifunctional crosslinker that facilitates thiol-maleimide reaction and inverse electron-demand Diels-Alder reaction (IEDDA). As a model protein, we selected 4D5scFv, derived from trastuzumab, a therapeutic antibody used in human epithermal growth factor 2 (HER2)-positive breast cancer treatment. We introduced a phenylalanine analog containing a very reactive tetrazine group (frTet) at conjugation site candidates predicted by computational methods. Using the linker TCO-PEG4-MAL, a single HSA molecule was site-specifically conjugated to the 4D5scFv (4D5scFv-HSA). The 4D5scFv-HSA conjugate exhibited HER2 binding affinity comparable to that of unmodified 4D5scFv. Furthermore, in pharmacokinetic profile in mice, the serum half-life of 4D5scFv-HSA was approximately 12 h, which is 85 times longer than that of 4D5scFv.ConclusionsThe antigen binding results and pharmacokinetic profile of 4D5scFv-HSA demonstrate that the site-specifically albumin-conjugated scFv retained its binding affinity with a prolonged serum half-life. In conclusion, we developed an effective strategy to prepare site-specifically albumin-conjugated 4D5scFv, which can have versatile clinical applications with improved efficacy.

An Inner Mitochondrial Membrane Microprotein from the SLC35A4 Upstream ORF Regulates Cellular Metabolism

Upstream open reading frames (uORFs) are cis-acting elements that can dynamically regulate the translation of downstream ORFs by suppressing downstream translation under basal conditions and, in some cases, increasing downstream translation under stress conditions. Computational and empirical methods have identified uORFs in the 5′-UTRs of approximately half of all mouse and human transcripts, making uORFs one of the largest regulatory elements known. Because the prevailing dogma was that eukaryotic mRNAs produce a single functional protein, the peptides and small proteins, or microproteins, encoded by uORFs were rarely studied. We hypothesized that a uORF in the SLC35A4 mRNA is producing a functional microprotein (SLC35A4-MP) because of its conserved amino acid sequence. Through a series of biochemical and cellular experiments, we find that the 103-amino acid SLC35A4-MP is a single-pass transmembrane inner mitochondrial membrane (IMM) microprotein. The IMM contains the protein machinery crucial for cellular respiration and ATP generation, and loss of function studies with SLC35A4-MP significantly diminish maximal cellular respiration, indicating a vital role for this microprotein in cellular metabolism. The findings add SLC35A4-MP to the growing list of functional microproteins and, more generally, indicate that uORFs that encode conserved microproteins are an untapped reservoir of functional microproteins.

Differentiation and functioning of the lateral line organ in zebrafish require Smpx activity

The small muscle protein, X-linked (SMPX) gene encodes a cytoskeleton-associated protein, highly expressed in the inner ear hair cells (HCs), possibly regulating auditory function. In the last decade, several mutations in SMPX have been associated with X-chromosomal progressive non syndromic hearing loss in humans and, in line with this, Smpx-deficient animal models, namely zebrafish and mouse, showed significant impairment of inner ear HCs development, maintenance, and functioning. In this work, we uncovered smpx expression in the neuromast mechanosensory HCs of both Anterior and Posterior Lateral Line (ALL and PLL, respectively) of zebrafish larvae and focused our attention on the PLL. Smpx was subcellularly localized throughout the cytoplasm of the HCs, as well as in their primary cilium. Loss-of-function experiments, via both morpholino-mediated gene knockdown and CRISPR/Cas9 F0 gene knockout, revealed that the lack of Smpx led to fewer properly differentiated and functional neuromasts, as well as to a smaller PLL primordium (PLLp), the latter also Smpx-positive. In addition, the kinocilia of Smpx-deficient neuromast HCs appeared structurally and numerically altered. Such phenotypes were associated with a significant reduction in the mechanotransduction activity of the neuromast HCs, in line with their positivity for Smpx. In summary, this work highlights the importance of Smpx in lateral line development and, specifically, in proper HCs differentiation and/or maintenance, and in the mechanotransduction process carried out by the neuromast HCs. Because lateral line HCs are both functionally and structurally analogous to the cochlear HCs, the neuromasts might represent an invaluable—and easily accessible—tool to dissect the role of Smpx in HCs development/functioning and shed light on the underlying mechanisms involved in hearing loss.

Improving prime editing with an endogenous small RNA-binding protein

Prime editing enables the precise modification of genomes through reverse transcription of template sequences appended to the 3′ ends of CRISPR–Cas guide RNAs1. To identify cellular determinants of prime editing, we developed scalable prime editing reporters and performed genome-scale CRISPR-interference screens. From these screens, a single factor emerged as the strongest mediator of prime editing: the small RNA-binding exonuclease protection factor La. Further investigation revealed that La promotes prime editing across approaches (PE2, PE3, PE4 and PE5), edit types (substitutions, insertions and deletions), endogenous loci and cell types but has no consistent effect on genome-editing approaches that rely on standard, unextended guide RNAs. Previous work has shown that La binds polyuridine tracts at the 3′ ends of RNA polymerase III transcripts2. We found that La functionally interacts with the 3′ ends of polyuridylated prime editing guide RNAs (pegRNAs). Guided by these results, we developed a prime editor protein (PE7) fused to the RNA-binding, N-terminal domain of La. This editor improved prime editing with expressed pegRNAs and engineered pegRNAs (epegRNAs), as well as with synthetic pegRNAs optimized for La binding. Together, our results provide key insights into how prime editing components interact with the cellular environment and suggest general strategies for stabilizing exogenous small RNAs therein.

Genome comparisons reveal accessory genes crucial for the evolution of apple Glomerella leaf spot pathogenicity in Colletotrichum fungi.

Apple Glomerella leaf spot (GLS) is an emerging fungal disease caused by Colletotrichum fructicola and other Colletotrichum species. These species are polyphyletic and it is currently unknown how these pathogens convergently evolved to infect apple. We generated chromosome-level genome assemblies of a GLS-adapted isolate and a non-adapted isolate in C. fructicola using long-read sequencing. Additionally, we resequenced 17 C. fructicola and C. aenigma isolates varying in GLS pathogenicity using short-read sequencing. Genome comparisons revealed a conserved bipartite genome architecture involving minichromosomes (accessory chromosomes) shared by C.fructicola and other closely related species within the C. gloeosporioides species complex. Moreover, two repeat-rich genomic regions (1.61 Mb in total) were specifically conserved among GLS-pathogenic isolates in C. fructicola and C. aenigma. Single-gene deletion of 10 accessory genes within the GLS-specific regions of C. fructicola identified three that were essential for GLS pathogenicity. These genes encoded a putative non-ribosomal peptide synthetase, a flavin-binding monooxygenase and a small protein with unknown function. These results highlight the crucial role accessory genes play in the evolution of Colletotrichum pathogenicity and imply the significance of an unidentified secondary metabolite in GLS pathogenesis.

Structural and Functional Impacts of Extended N-Terminal End of the Small Heat Shock Protein Tpv HSP 14.3

Structural and Functional Impacts of Extended N-Terminal End of the Small Heat Shock Protein Tpv HSP 14.3

ArHsp90 is important in stress tolerance and embryo development of the brine shrimp, Artemia franciscana.

Females of the extremophile crustacean, Artemia franciscana, either release motile nauplii via the ovoviviparous pathway or encysted embryos (cysts) via the oviparous pathway. Cysts contain an abundant amount of the ATP-independent small heat shock protein that contributes to stress tolerance and embryo development, however, little is known of the role of ATP-dependent molecular chaperone, heat shock protein 90 (Hsp90) in the two processes. In this study, a hsp90 was cloned from A.franciscana. Characteristic domains of ArHsp90 were simulated from the deduced amino acid sequence, and 3D structures of ArHsp90 and Hsp90s of organisms from different groups were aligned. RNA interference was then employed to characterize ArHsp90 in A.franciscana nauplii and cysts. The partial knockdown of ArHsp90 slowed the development of nauplius-destined, but not cyst-destined embryos. ArHsp90 knockdown also reduced the survival and stress tolerance of nauplii newly released from A.franciscana females. Although the reduction of ArHsp90 had no effect on the development of diapause-destined embryos, the resulting cysts displayed reduced tolerance to desiccation and low temperature, two stresses normally encountered by A.franciscana in its natural environment. The results reveal that Hsp90 contributes to the development, growth, and stress tolerance of A.franciscana, an organism of practical importance as a feed source in aquaculture.

Capsicum chinense Jacq.-derived glutaredoxin (CcGRXS12) alters redox status of the cells to confer resistance against pepper mild mottle virus (PMMoV-I).

The CcGRXS12 gene protects plants from cellular oxidative damage that are caused by both biotic and abiotic stresses. The protein possesses GSH-disulphide oxidoreductase property but lacks Fe-S cluster assembly mechanism. Glutaredoxins (Grxs) are small, ubiquitous and multi-functional proteins. They are present in different compartments of plant cells. A chloroplast targeted Class I GRX (CcGRXS12) gene was isolated from Capsicum chinense during the pepper mild mottle virus (PMMoV) infection. Functional characterization of the gene was performed in Nicotiana benthamiana transgenic plants transformed with native C. chinense GRX (Nb:GRX), GRX-fused with GFP (Nb:GRX-GFP) and GRX-truncated for chloroplast sequences fused with GFP (Nb:Δ2MGRX-GFP). Overexpression of CcGRXS12 inhibited the PMMoV-I accumulation at the later stage of infection, accompanied with the activation of salicylic acid (SA) pathway pathogenesis-related (PR) transcripts and suppression of JA/ET pathway transcripts. Further, the reduced accumulation of auxin-induced Glutathione-S-Transferase (pCNT103) in CcGRXS12 overexpressing lines indicated that the protein could protect the plants from the oxidative stress caused by the virus. PMMoV-I infection increased the accumulation of pyridine nucleotides (PNs) mainly due to the reduced form of PNs (NAD(P)H), and it was high in Nb:GRX-GFP lines compared to other transgenic lines. Apart from biotic stress, CcGRXS12 protects the plants from abiotic stress conditions caused by H2O2 and herbicide paraquat. CcGRXS12 exhibited GSH-disulphide oxidoreductase activity in vitro; however, it was devoid of complementary Fe-S cluster assembly mechanism found in yeast. Overall, this study proves that CcGRXS12 plays a crucial role during biotic and abiotic stress in plants.