Unusual 1-3 peptidoglycan cross-links in Acetobacteraceae are made by L,D-transpeptidases with a catalytic domain distantly related to YkuD domains.

Peptidoglycan is an essential component of the bacterial cell envelope that contains glycan chains substituted by short peptide stems. Peptide stems are polymerized by D,D-transpeptidases, which make bonds between the amino acid in position four of a donor stem and the third residue of an acceptor stem (4-3 cross-links). Some bacterial peptidoglycans also contain 3-3 cross-links that are formed by another class of enzymes called L,D-transpeptidases which contain a YkuD catalytic domain. In this work, we investigate the formation of unusual bacterial 1-3 peptidoglycan cross-links. We describe a version of the PGFinder software that can identify 1-3 cross-links and report the high-resolution peptidoglycan structure of Gluconobacter oxydans (a model organism within the Acetobacteraceae family). We reveal that G. oxydans peptidoglycan contains peptide stems made of a single alanine as well as several dipeptide stems with unusual amino acids at their C-terminus. Using a bioinformatics approach, we identified a G. oxydans mutant from a transposon library with a drastic reduction in 1-3 cross-links. Through complementation experiments in G. oxydans and recombinant protein production in a heterologous host, we identify an L,D-transpeptidase enzyme with a domain distantly related to the YkuD domain responsible for these non-canonical reactions. This work revisits the enzymatic capabilities of L,D-transpeptidases, a versatile family of enzymes that play a key role in bacterial peptidoglycan remodelling.

Expression of human BRCA2 in Saccharomyces cerevisiae complements the loss of RAD52 in double-strand break repair.

BRCA2 is a tumor-suppressor gene that is normally expressed in the breast and ovarian tissue of mammals. The BRCA2 protein mediates the repair of double-strand breaks (DSBs) using homologous recombination, which is a conserved pathway in eukaryotes. Women who express missense mutations in the BRCA2 gene are predisposed to an elevated lifetime risk for both breast cancer and ovarian cancer. In the present study, the efficiency of human BRCA2 (hBRCA2) in DSB repair was investigated in the budding yeast Saccharomyces cerevisiae. While budding yeast does not possess a true BRCA2 homolog, they have a potential functional homolog known as Rad52, which is an essential repair protein involved in mediating homologous recombination using the same mechanism as BRCA2 in humans. Therefore, to examine the functional overlap between Rad52 in yeast and hBRCA2, we expressed the wild-type hBRCA2 gene in budding yeast with or without Rad52 and monitored ionizing radiation resistance and DSB repair efficiency. We found that the expression of hBRCA2 in rad52 mutants increases both radiation resistance and DSB repair frequency compared to cells not expressing BRCA2. Specifically, BRCA2 improved the protection against ionizing radiation by at least 1.93-fold and the repair frequency by 6.1-fold. In addition, our results show that homology length influences repair efficiency in rad52 mutant cells, which impacts BRCA2 mediated repair of DSBs. This study provides evidence that S. cerevisiae could be used to monitor BRCA2 function, which can help in understanding the genetic consequences of BRCA2 variants and how they may contribute to cancer progression.

Expanded in vivo substrate profile of the yeast N-terminal acetyltransferase NatC.

N-terminal acetylation is a conserved protein modification among eukaryotes. The yeast Saccharomyces cerevisiae is a valuable model system for studying this modification. The bulk of protein N-terminal acetylation in S. cerevisiae is catalyzed by the N-terminal acetyltransferases NatA, NatB, and NatC. Thus far, proteome-wide identification of the in vivo protein substrates of yeast NatA and NatB has been performed by N-terminomics. Here, we used S. cerevisiae deleted for the NatC catalytic subunit Naa30 and identified 57 yeast NatC substrates by N-terminal combined fractional diagonal chromatography analysis. Interestingly, in addition to the canonical N-termini starting with ML, MI, MF, and MW, yeast NatC substrates also included MY, MK, MM, MA, MV, and MS. However, for some of these substrate types, such as MY, MK, MV, and MS, we also uncovered (residual) non-NatC NAT activity, most likely due to the previously established redundancy between yeast NatC and NatE/Naa50. Thus, we have revealed a complex interplay between different NATs in targeting methionine-starting N-termini in yeast. Furthermore, our results showed that ectopic expression of human NAA30 rescued known NatC phenotypes in naa30Δ yeast, as well as partially restored the yeast NatC Nt-acetylome. Thus, we demonstrate an evolutionary conservation of NatC from yeast to human thereby underpinning future disease models to study pathogenic NAA30 variants. Overall, this work offers increased biochemical and functional insights into NatC-mediated N-terminal acetylation and provides a basis for future work to pinpoint the specific molecular mechanisms that link the lack of NatC-mediated N-terminal acetylation to phenotypes of NatC deletion yeast.

A mechanism for oxidative damage repair at gene regulatory elements.

Oxidative genome damage is an unavoidable consequence of cellular metabolism. It arises at gene regulatory elements by epigenetic demethylation during transcriptional activation1,2. Here we show that promoters are protected from oxidative damage via a process mediated by the nuclear mitotic apparatus protein NuMA (also known as NUMA1). NuMA exhibits genomic occupancy approximately 100 bp around transcription start sites. It binds the initiating form of RNA polymerase II, pause-release factors and single-strand break repair (SSBR) components such as TDP1. The binding is increased on chromatin following oxidative damage, and TDP1 enrichment at damaged chromatin is facilitated by NuMA. Depletion of NuMA increases oxidative damage at promoters. NuMA promotes transcription by limiting the polyADP-ribosylation of RNA polymerase II, increasing its availability and release from pausing at promoters. Metabolic labelling of nascent RNA identifies genes that depend on NuMA for transcription including immediate-early response genes. Complementation of NuMA-deficient cells with a mutant that mediates binding to SSBR, or a mitotic separation-of-function mutant, restores SSBR defects. These findings underscore the importance of oxidative DNA damage repair at gene regulatory elements and describe a process that fulfils this function.

A novel kinase subverts aluminium resistance by boosting ornithine decarboxylase-dependent putrescine biosynthesis.

Rice, as one of the most aluminium (Al)-resistant cereal crops, has developed more complicated Al resistance mechanisms than others. By using forward genetic screening from a rice ethyl methanesulfonate mutant library, we obtained a mutant showing specifically high sensitivity to Al. Through MutMap analysis followed by a complementation test, we identified the causal gene, Al-related Protein Kinase (ArPK) for Al-sensitivity. ArPK expression was induced by a relatively longer exposure to high Al concentration in the roots. The result of RNA-sequencing indicated the functional disorder in arginine metabolism pathway with downregulation of N-acetylornithine deacetylase (NAOD) expression and upregulation of Ornithine decarboxylase1 (ODC1) expression in arpk mutant. Al specifically and rapidly upregulated ODC1 expression and causes overaccumulation of putrescine (Put), whereas the ODC inhibitor difluoromethylornithine reverted Al-sensitive phenotype of arpk, suggesting that overaccumulation of endogenous Put might be harmful for root growth, and that ArPK seems to act as an endogenous inhibitor of ODC1 action to maintain suitable endogenous Put level under Al treatment. Overall, we identified ArPK and its putative repressive role in controlling a novel ODC-dependent Put biosynthesis pathway specifically affecting rice Al resistance, thus enriching the fundamental understanding of plant Al resistance.

Whole genome resequencing and complementation tests reveal candidate loci contributing to bacterial wilt (Ralstonia sp.) resistance in tomato.

Tomato (Solanum lycopersicum) is one of the most economically important vegetable crops worldwide. Bacterial wilt (BW), caused by the Ralstonia solanacearum species complex, has been reported as the second most important plant pathogenic bacteria worldwide, and likely the most destructive. Extensive research has identified two major loci, Bwr-6 and Bwr-12, that contribute to resistance to BW in tomato; however, these loci do not completely explain resistance. Segregation of resistance in two populations that were homozygous dominant or heterozygous for all Bwr-6 and Bwr-12 associated molecular markers suggested the action of one or two resistance loci in addition to these two major QTLs. We utilized whole genome sequence data analysis and pairwise comparison of six BW resistant and nine BW susceptible tomato lines to identify candidate genes that, in addition to Bwr-6 and Bwr-12, contributed to resistance. Through this approach we found 27,046 SNPs and 5975 indels specific to the six resistant lines, affecting 385 genes. One sequence variant on chromosome 3 captured by marker Bwr3.2dCAPS located in the Asc (Solyc03g114600.4.1) gene had significant association with resistance, but it did not completely explain the resistance phenotype. The SNP associated with Bwr3.2dCAPS was located within the resistance gene Asc which was inside the previously identified Bwr-3 locus. This study provides a foundation for further investigations into new loci distributed throughout the tomato genome that could contribute to BW resistance and into the role of resistance genes that may act against multiple pathogens.

Détection de la mutation T790M de l'exon 20 du gène EGFR dans le cancer du poumon résistant aux inhibiteurs de l'EGFR sur ADN tumoral circulant (biopsie liquide) / Circulating tumour DNA (liquid biopsy)-based EGFR exon 20 T790M mutation detection in advanced non-small cell lung cancer after previous EGFR-directed therapy

DEMANDEUR: Institut universitaire de cardiologie et de pneumologie de Québec Université Laval. OBJECTIF DE L'ANALYSE: L'analyse proposée vise à effectuer la détection de la mutation T790M du gène EGFR (EGFR-T790M) responsable de la résistance aux inhibiteurs de tyrosine kinase (ITK) à partir de l'ADN tumoral circulant d'un patient atteint d'un cancer du poumon non à petites cellules (CPNPC). CONTEXTE DE LA DEMANDE: Approximativement 60 % des patients atteints d'un CPNPC et traités avec un ITK de 1re ou de 2e génération vont développer la mutation de résistance EGFR-T790M et avoir besoin d'un changement thérapeutique. Bien qu'une analyse permettant notamment de détecter cette mutation à partir d'une biopsie tissulaire soit consignée dans le Répertoire québécois et système de mesure des procédures de biologie médicale (ci-après nommé « Répertoire ¼), la présente demande vise à détecter la mutation EGFR-T790M à partir d'échantillons de biopsie liquide, une autre méthode de prélèvement qui permet au patient d'éviter la biopsie tissulaire, les risques qui y sont associés et d'obtenir un changement thérapeutique plus rapidement. NOMBRE D'ANALYSES PRÉVUES: Le demandeur avait originalement prévu que, annuellement, entre 160 et 250 tests seraient nécessaires pour servir la population locale et 2 000 pour l'ensemble du Québec. Or, l'inscription de l'osimertinib en 1re intention de traitement des CPNPC avec mutations activatrices de l'EGFR entraîne une réduction importante du nombre de patients dont l'état nécessitera éventuellement une détection de la mutation EGFRT790M par biopsie liquide selon l'utilisation prévue par le demandeur. Les données de la RAMQ ont révélé que quelques patients continuent de recevoir des ITK de 1re et de 2e génération et sont susceptibles de tirer avantage de cette analyse. MÉTHODOLOGIE: La démarche d'évaluation comprend une revue rapide de la littérature scientifique et grise de même que des consultations menées auprès d'experts et d'autres parties prenantes. Un rapport d'évaluation portant sur la pertinence de recourir à la biopsie liquide dans le même contexte que la présente demande a été publié par Health Quality Ontario (HQO) en mars 2020. Ce rapport comporte une évaluation de la performance diagnostique, de l'utilité clinique, de la sécurité, de l'efficience et de l'impact budgétaire de même qu'une analyse des préférences et des valeurs selon la perspective du patient. Le rapport de HQO a été jugé de bonne qualité méthodologique et constitue la principale source de données de la présente évaluation. L'ensemble des données scientifiques, contextuelles et expérientielles a été interprété et apprécié à l'aide d'une grille-synthèse pour guider le processus de délibération du Comité scientifique des analyses de biologie médicale (CSABM). DONNÉES PUBLIÉES: Performance diagnostique: Selon le rapport d'évaluation de Health Quality Ontario publié en 2020, la concordance des résultats d'une détection de la mutation EGFRT790M entre la biopsie liquide et la biopsie tissulaire varierait de 50 % à 96 %. La sensibilité et la spécificité, la valeur prédictive négative (VPN) et la valeur prédictive positive (VPP) sont respectivement de 68 % [46 % - 88 %], 86 % [62 % - 99 %], 61 % et 89 %. L'utilisation de la droplet digital PCR (ddPCR) augmenterait la fréquence de détection positive de la mutation EGFR-T790M par biopsie liquide. Utilité clinique: HQO n'a repéré aucune donnée sur l'utilisation de la biopsie liquide comme méthode de triage (biopsie liquide + biopsie tissulaire) comparativement à la biopsie tissulaire seule. Toutefois, les études consultées soulignent qu'une fraction des porteurs de la mutation EGFR-T790M (17,8 %) sont identifiés grâce à l'utilisation de la biopsie liquide pour trier les patients résistants aux inhibiteurs des ITK de 1re et 2e génération et pour lesquels la biopsie tissulaire est évitée. En cas de détection de la mutation EGFR-T790M, l'impact sur la prise en charge demeurerait le même quelle que soit la méthode employée car, lorsque la biopsie est positive, le traitement est amorcé. Un gain de survie sans progression de 9,7 mois a été rapporté pour ces deux méthodes. PERSPECTIVE DES PATIENTS: Selon le rapport de HQO, les patients percevraient la biopsie liquide comme une approche plus rapide et plus pratique du fait qu'ils n'auraient pas à attendre plusieurs semaines pour obtenir un rendezvous pour subir une biopsie tissulaire qui pourrait nécessiter un déplacement vers un centre spécialisé plus éloigné. Ils auraient aussi exprimé de la peur et même de la panique relativement au processus de prélèvement de l'échantillon tissulaire qui nécessite une aiguille de gros calibre. POSITIONS ET ORIENTATIONS D'ORGANISMES D'INTÉRÊ: Le National Comprehensive Cancer Network (NCCN) et plusieurs comités d'experts ont recommandé l'utilisation de la biopsie liquide chez un patient qui est médicalement incapable de supporter l'échantillonnage effractif que requiert la biopsie tissulaire. L'ensemble des sociétés savantes recommandent l'utilisation de la biopsie tissulaire lorsque le résultat de la biopsie liquide est négatif. Toutefois, ils soulignent que la biopsie liquide ne devrait pas remplacer la biopsie tissulaire. En revanche, un résultat positif de la biopsie liquide devrait obtenir la même attention qu'un résultat positif de la biopsie tissulaire. ÉVALUATION ÉCONOMIQUE: La biopsie liquide comme méthode de triage est moins coûteuse et plus efficace que la biopsie tissulaire pour détecter la mutation EGFRT790M. Toutefois, lorsqu'on tient compte des avantages cliniques et des coûts liés à l'usage des traitements subséquents, le ratio coûtutilité incrémental est élevé, en raison notamment de l'inefficience de l'osimertinib. Depuis l'inscription aux listes des médicaments de l'osimertinib comme traitement de 1re intention, la population admissible à une biopsie liquide décroît continuellement. Le nombre actuel de personnes qui recevraient des ITK de 1re et 2e génération a été estimé à 32 selon les données de la RAMQ et à 40 en incluant les patients couverts par une assurance privée. Ces analyses estiment les économies de laboratoire à 460 $, mais elles anticipent que 5 patients supplémentaires pourraient recevoir l'osimertinib pour un impact budgétaire net d'environ 537 000 $ au cours des trois prochaines années. POSITION DES EXPERTS CONSULTÉS: Les experts consultés sont favorables à l'utilisation de la biopsie liquide dans le contexte décrit par le demandeur. Ils n'ont pas soulevé d'enjeu en particulier en dehors de la pertinence restreinte de cette analyse étant donné l'inscription récente du traitement osimertinib en 1re intention. L'émergence rapide de nombreuses indications de la biopsie liquide serait imminente et constituerait une révolution technologique et oncologique. ENJEUX PARTICULIERS: Au Québec, l'analyse est principalement employée en contexte de recherche. Des enjeux éthiques et cliniques liés à la biopsie liquide tels que la présence de faux négatifs en raison d'une sensibilité plus faible et la nécessité d'évaluer la survie globale des patients atteints de CPNPC en appliquant cette technique ont également été rapportés dans la littérature. RECOMMANDATION: Suivant une délibération par les membres du CSABM sur l'ensemble de la preuve, y compris la perspective des experts consultés, l'INESSS recommande d'introduire la détection de la mutation EGFR-T790M sur ADN tumoral circulant au Répertoire.

REQUESTER: Institut universitaire de cardiologie et de pneumologie de QuébecUniversité Laval. PURPOSE OF TEST: The test is designed to detect the EGFR gene T790M mutation (EGFRT790M), which is responsible for resistance to tyrosine kinase inhibitors (TKIs), in circulating tumour DNA in non-small cell lung cancer (NSCLC) patients. BACKGROUND TO THE REQUEST: Approximately 60% of NSCLC patients treated with a 1st- or 2ndgeneration TKI will develop the EGFR-T790M resistance mutation and require a change in therapy. Although a test used to detect this mutation from a tissue biopsy is listed in the Répertoire québécois et système de mesure des procédures de biologie médicale (hereinafter the "Répertoire"), the present request concerns the detection of the EGFRT790M mutation in samples obtained by liquid biopsy, an alternative specimen collection method that enables the patient to avoid a tissue biopsy and the associated risks, and to obtain a change in therapy more quickly. EXPECTED NUMBER OF TESTS: The requester had originally anticipated that between 160 and 250 tests would be required annually to serve the local population and 2000 for all of Québec. However, the listing of osimertinib as a 1st-line therapy for NSCLC with EGFR-activating mutations is resulting in a significant reduction in the number of patients who will possibly require EGFRT790M mutation testing by liquid biopsy, based on the use anticipated by the requester. RAMQ data show that a few patients continue to receive 1st- or 2nd-generation TKIs and are likely to benefit from this test. METHODOLOGY: The assessment process included a rapid review of the scientific and grey literature, and consultations with experts and other stakeholders. An assessment report on the relevance of using liquid biopsy in the same context as this request was published by Health Quality Ontario (HQO) in March 2020. The report includes an assessment of its diagnostic performance, clinical utility, safety, cost-effectiveness and budget impact, and an assessment of patient preferences and values. The HQO report was deemed to be of good methodological quality and is the main source of data for the present assessment. All the scientific, contextual and experiential data were interpreted and assessed using a synthesis grid to guide the Comité scientifique des analyses de biologie médicale (CSABM)'s deliberation process. PUBLISHED DATA: Diagnostic performance: According to the Health Quality Ontario assessment report published in 2020, the concordance rate for EGFR-T790M mutation detection results between liquid biopsy and tissue biopsy ranges from 50% to 96%. The sensitivity and specificity, negative predictive value (NPV) and positive predictive value (PPV) are 68% [46%-88%], 86% [62%-99%], 61% and 89%, respectively. The use of droplet digital PCR (ddPCR) appears to increase the rate of positive detection of the EGFR-T790M mutation by liquid biopsy. Clinical utility: HQO did not find any data on the use of liquid biopsy as a triage test (liquid biopsy + tissue biopsy) versus tissue biopsy alone. However, the studies examined note that a proportion of EGFR-T790M mutation carriers (17.8%) are identified through the use of liquid biopsy to detect 1st- or 2nd-generation TKI-resistant patients, who thus avoid a tissue biopsy. If the EGFR-T790M mutation is detected, the impact on management would remain the same, regardless of the method used, because when the biopsy is positive, treatment is initiated. A 9.7-month gain in progression-free survival was reported for both methods. PATIENT PERSPECTIVE: According to the HQO report, patients perceived liquid biopsy as a faster and more convenient approach because they would not have to wait several weeks to get an appointment for a tissue biopsy, which could require a trip to a specialized centre that might be further away. They also expressed fear and even panic about the tissue sample collection procedure, in which a large-gauge needle is used. POSITIONS AND PERSPECTIVES OF ORGANIZATIONS OF INTEREST: The National Comprehensive Cancer Network (NCCN) and a number of expert panels have recommended the use of liquid biopsy in patients who are medically unable to tolerate the invasive specimen collection involved in a tissue biopsy. All the learned societies recommend the use of tissue biopsy when the liquid biopsy result is negative. However, they stress that liquid biopsy should not replace tissue biopsy. In any event, a positive liquid biopsy result should be given the same attention as a positive tissue biopsy result. ECONOMIC EVALUATION: Liquid biopsy as a triage test is less expensive and more effective than tissue biopsy in detecting the EGFR-T790M mutation. However, when the clinical benefits and costs of the subsequent treatments are factored. in, the incremental cost-utility ratio is high, in part because of osimertinib's non-cost-effectiveness. Since the listing of this drug as a 1st-line therapy in the formularies, the liquid biopsy-eligible population has been steadily decreasing. The current number of people receiving 1st- or 2nd-generation TKIs was estimated to be 32, based on RAMQ data, and 40 if patients with private insurance are included. These analyses estimate the laboratory savings at $460, but they anticipate that 5 additional patients could receive osimertinib, for a net budget impact of approximately $537,000 over the next 3 years. POSITION OF THE EXPERTS CONSULTED: The experts consulted support the use of liquid biopsy in the context defined by the requester. They did not raise any specific issues, apart from the limited relevance of this test, given the recent listing of osimertinib as a 1st-line therapy. The rapid emergence of a large number of indications for liquid biopsy appears to be imminent and would constitute a technological and oncologic revolution. SPECIFIC ISSUES: In Québec, liquid biopsy is used primarily in research settings. Ethical and clinical issues associated with this test, such as false negatives due to lower sensitivity and the need to assess overall survival in NSCLC patients in whom this technique is used, have also been reported in the literature. RECOMMENDATION: Based on the deliberation, by the CSABM's members, of all the evidence, including the perspective of the experts consulted, INESSS recommends that circulating tumour DNA-based EGFR-T790M mutation detection be included in the Répertoire

Recombinant Lloviu virus as a tool to study viral replication and host responses.

Next generation sequencing has revealed the presence of numerous RNA viruses in animal reservoir hosts, including many closely related to known human pathogens. Despite their zoonotic potential, most of these viruses remain understudied due to not yet being cultured. While reverse genetic systems can facilitate virus rescue, this is often hindered by missing viral genome ends. A prime example is Lloviu virus (LLOV), an uncultured filovirus that is closely related to the highly pathogenic Ebola virus. Using minigenome systems, we complemented the missing LLOV genomic ends and identified cis-acting elements required for LLOV replication that were lacking in the published sequence. We leveraged these data to generate recombinant full-length LLOV clones and rescue infectious virus. Similar to other filoviruses, recombinant LLOV (rLLOV) forms filamentous virions and induces the formation of characteristic inclusions in the cytoplasm of the infected cells, as shown by electron microscopy. Known target cells of Ebola virus, including macrophages and hepatocytes, are permissive to rLLOV infection, suggesting that humans could be potential hosts. However, inflammatory responses in human macrophages, a hallmark of Ebola virus disease, are not induced by rLLOV. Additional tropism testing identified pneumocytes as capable of robust rLLOV and Ebola virus infection. We also used rLLOV to test antivirals targeting multiple facets of the replication cycle. Rescue of uncultured viruses of pathogenic concern represents a valuable tool in our arsenal for pandemic preparedness.

Molecular cloning and functional characterization of UBC13 and MMS2 from Candida albicans.

To maintain genome stability, eukaryotes have evolved a powerful DNA damage response system called DNA-damage tolerance (DDT) to deal with replication-blocking lesions. In the budding yeast Saccharomyces cerevisiae, K63-linked polyubiquitination of proliferating cell nuclear antigen (PCNA) is mediated by a Ubc13-Mms2 heterodimer, leading to error-free DDT. Candida albicans is one of the most studied fungal pathogens and to date no data regarding K63-linked ubiquitination or error-free DDT has been available. Here we report the identification and functional characterization of UBC13 and MMS2 genes from C. albicans. Both genes are highly conserved between S. cerevisiae and C. albicans. However, CaUbc13 differs from all other eukaryotes in that it contains a 21-amino acid tail that appears to attenuate its interaction with CaMms2, suggesting a possible regulatory mechanism in C. albicans. Both CaUBC13 and CaMMS2 genes can functionally rescue the corresponding budding yeast mutants from increased spontaneous mutagenesis and killing by DNA-damaging agents, indicating an error-free DDT pathway in C. albicans. Indeed Caubc13Δ/Δ and Camms2Δ/Δ null mutants were constructed and displayed characteristic sensitivity to DNA-damaging agents.

Interspecific complementation-restoration of phenotype in Arabidopsis cuc2cuc3 mutant by sugarcane CUC2 gene.

BACKGROUND: In plants, a critical balance between differentiation and proliferation of stem cells at the shoot apical meristem zone is essential for proper growth. The spatiotemporal regulation of some crucial genes dictates the formation of a boundary within and around budding organs. The boundary plays a pivotal role in distinguishing one tissue type from another and provides a defined shape to the organs at their developed stage. NAM/CUC subfamily of the NAC transcription factors control the boundary formation during meristematic development. RESULTS: Here, we have identified the CUP-SHAPED COTYLEDON (CUC) genes in sugarcane and named SsCUC2 (for the orthologous gene of CUC1 and CUC2) and SsCUC3. The phylogenetic reconstruction showed that SsCUCs occupy the CUC2 and CUC3 clade together with monocots, whereas eudicot CUC2 and CUC3 settled separately in the different clade. The structural analysis of CUC genes showed that most of the CUC3 genes were accompanied by an intron gain during eudicot divergence. Besides, the study of SsCUCs expression in the RNA-seq obtained during different stages of ovule development revealed that SsCUCs express in developing young tissues, and the expression of SsCUC2 is regulated by miR164. We also demonstrate that SsCUC2 (a monocot) could complement the cuc2cuc3 mutant phenotype of Arabidopsis (eudicot). CONCLUSIONS: This study further supports that CUC2 has diverged in CUC1 and CUC2 during the evolution of monocots and eudicots from ancestral plants. The functional analysis of CUC expression patterns during sugarcane ovule development and ectopic expression of SsCUC2 in Arabidopsis showed that SsCUC2 has a conserved role in boundary formation. Overall, these findings improve our understanding of the functions of sugarcane CUC genes. Our results reveal the crucial functional role of CUC genes in sugarcane.

Genetic and Physiological Characterization of Fructose-1,6-Bisphosphate Aldolase and Glyceraldehyde-3-Phosphate Dehydrogenase in the Crabtree-Negative Yeast Kluyveromyces lactis.

The milk yeast Kluyveromyces lactis degrades glucose through glycolysis and the pentose phosphate pathway and follows a mainly respiratory metabolism. Here, we investigated the role of two reactions which are required for the final steps of glucose degradation from both pathways, as well as for gluconeogenesis, namely fructose-1,6-bisphosphate aldolase (FBA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In silico analyses identified one gene encoding the former (KlFBA1), and three genes encoding isoforms of the latter (KlTDH1, KlTDH2, KlGDP1). Phenotypic analyses were performed by deleting the genes from the haploid K. lactis genome. While Klfba1 deletions lacked detectable FBA activity, they still grew poorly on glucose. To investigate the in vivo importance of the GAPDH isoforms, different mutant combinations were analyzed for their growth behavior and enzymatic activity. KlTdh2 represented the major glycolytic GAPDH isoform, as its lack caused a slower growth on glucose. Cells lacking both KlTdh1 and KlTdh2 failed to grow on glucose but were still able to use ethanol as sole carbon sources, indicating that KlGdp1 is sufficient to promote gluconeogenesis. Life-cell fluorescence microscopy revealed that KlTdh2 accumulated in the nucleus upon exposure to oxidative stress, suggesting a moonlighting function of this isoform in the regulation of gene expression. Heterologous complementation of the Klfba1 deletion by the human ALDOA gene renders K. lactis a promising host for heterologous expression of human disease alleles and/or a screening system for specific drugs.

Structure-Function Analysis of Two Interacting Vaccinia Proteins That Are Critical for Viral Morphogenesis: L2 and A30.5.

An enduring mystery in poxvirology is the mechanism by which virion morphogenesis is accomplished. A30.5 and L2 are two small regulatory proteins that are essential for this process. Previous studies have shown that vaccinia A30.5 and L2 localize to the ER and interact during infection, but how they facilitate morphogenesis is unknown. To interrogate the relationship between A30.5 and L2, we generated inducible complementing cell lines (CV1-HA-L2; CV1-3xFLAG-A30.5) and deletion viruses (vΔL2; vΔA30.5). Loss of either protein resulted in a block in morphogenesis and a significant (>100-fold) decrease in infectious viral yield. Structure-function analysis of L2 and A30.5, using transient complementation assays, identified key functional regions in both proteins. A clustered charge-to-alanine L2 mutant (L2-RRD) failed to rescue a vΔL2 infection and exhibits a significantly retarded apparent molecular weight in vivo (but not in vitro), suggestive of an aberrant posttranslational modification. Furthermore, an A30.5 mutant with a disrupted putative N-terminal α-helix failed to rescue a vΔA30.5 infection. Using our complementing cell lines, we determined that the stability of A30.5 is dependent on L2 and that wild-type L2 and A30.5 coimmunoprecipitate in the absence of other viral proteins. Further examination of this interaction, using wild-type and mutant forms of L2 or A30.5, revealed that the inability of mutant alleles to rescue the respective deletion viruses is tightly correlated with a failure of L2 to stabilize and interact with A30.5. L2 appears to function as a chaperone-like protein for A30.5, ensuring that they work together as a complex during viral membrane biogenesis. IMPORTANCE Vaccinia virus is a large, enveloped DNA virus that was successfully used as the vaccine against smallpox. Vaccinia continues to be an invaluable biomedical research tool in basic research and in gene therapy vector and vaccine development. Although this virus has been studied extensively, the complex process of virion assembly, termed morphogenesis, still puzzles the field. Our work aims to better understand how two small viral proteins that are essential for viral assembly, L2 and A30.5, function during early morphogenesis. We show that A30.5 requires L2 for stability and that these proteins interact in the absence of other viral proteins. We identify regions in each protein required for their function and show that mutations in these regions disrupt the interaction between L2 and A30.5 and fail to restore virus viability.

Orchardgrass ACTIVATOR OF HSP90 ATPASE possesses autonomous chaperone properties and activates Hsp90 transcription to enhance thermotolerance.

High temperature stress is an environmental factor that negatively affects the growth and development of crops. Hsp90 (90 kDa heat shock protein) is a major molecular chaperone in eukaryotic cells, contributing to the maintenance of cell homeostasis through interaction with co-chaperones. Aha1 (activator of Hsp90 ATPase) is well known as a co-chaperone that activates ATPase activity of Hsp90 in mammals. However, biochemical and physiological evidence relating to Aha has not yet been identified in plants. In this study, we investigated the heat-tolerance function of orchardgrass (Dactylis glomerata L.) Aha (DgAha). Recombinant DgAha interacted with cytosolic DgHsp90s and efficiently protected substrates from thermal denaturation. Furthermore, heterologous expression of DgAha in yeast (Saccharomyces cerevisiae) cells and Arabidopsis (Arabidopsis thaliana) plants conferred thermotolerance in vivo. Enhanced expression of DgAha in Arabidopsis stimulates the transcription of Hsp90 under heat stress. Our data demonstrate that plant Aha plays a positive role in heat stress tolerance via chaperone properties and/or activation of Hsp90 to protect substrate proteins in plants from thermal injury.

A split NanoLuc complementation-based human norovirus-like particle entry assay facilitates evaluation of anti-norovirus antibodies in live cells.

Human noroviruses (NoVs) are the most common cause of acute gastroenteritis worldwide. One major obstacle in developing NoV vaccines is the lack of robust cell culture for efficacy evaluation. In this study, we successfully developed a NoV virus-like particle (VLP) entry assay based on split NanoLuc luciferase (LgBiT and HiBiT) complementation. HiBiT-tagged NoV GII.4 VLP (VLP-HiBiT) can be efficiently produced in Pichia pastoris and retain binding activity towards NoV receptor histo-blood group antigens (HBGAs). A 293T-FUT2-LgBiT cell line was established and was shown to stably express cell surface HBGAs and intracellular LgBiT. GII.4 VLP-HiBiT can bind and enter into the 293-FUT2-LgBiT cells, producing strong luminescence signals in live cells. Anti-GII.4 sera can inhibit VLP-HiBiT entry into the 293-FUT2-LgBiT cells in a dose-dependent manner, and neutralizing titers well correlate with their blocking titers measured by HBGAs-binding blockade assay. Moreover, such a surrogate infection/neutralization assay can be applied to other NoV genotypes such as GI.1 and GII.17. Together, the VLP-HiBiT entry assay can mimic both NoV attachment and internalization in live cells and thus facilitate reliable and comprehensive evaluation of NoV vaccine and antibodies.

Species-selective targeting of pathogens revealed by the atypical structure and active site of Trypanosoma cruzi histone deacetylase DAC2.

Writing and erasing of posttranslational modifications are crucial to phenotypic plasticity and antigenic variation of eukaryotic pathogens. Targeting pathogens' modification machineries, thus, represents a valid approach to fighting parasitic diseases. However, identification of parasitic targets and the development of selective anti-parasitic drugs still represent major bottlenecks. Here, we show that the zinc-dependent histone deacetylases (HDACs) of the protozoan parasite Trypanosoma cruzi are key regulators that have significantly diverged from their human counterparts. Depletion of T. cruzi class I HDACs tcDAC1 and tcDAC2 compromises cell-cycle progression and division, leading to cell death. Notably, tcDAC2 displays a deacetylase activity essential to the parasite and shows major structural differences with human HDACs. Specifically, tcDAC2 harbors a modular active site with a unique subpocket targeted by inhibitors showing substantial anti-parasitic effects in cellulo and in vivo. Thus, the targeting of the many atypical HDACs in pathogens can enable anti-parasitic selective chemical impairment.

Phenotypic effects from the expression of a deregulated AtGAD1 transgene and GABA pathway suppression mutants in maize.

Glutamate decarboxylase (GAD; EC 4.1.1.15) catalyzes the irreversible decarboxylation of glutamate to produce γ-aminobutyric acid (GABA); a ubiquitous non-protein amino acid involved in the regulation of several aspects of plant metabolism and physiology. To study the function of GAD and GABA in maize, we have; 1) introduced native and deregulated forms of AtGAD1 into maize with the intent of increasing the synthesis of GABA and 2) introduced constructs into maize designed to suppress the activity of several GABA shunt, GABA transport and GABA pathway genes. Maize plants expressing the deregulated AtGAD1 exhibit a severe chlorosis and retarded growth phenotype and have high levels of GABA, and Ca++/CaM-independent GAD activity. Plants expressing the suppression constructs for GABA biosynthetic and transport pathway genes had no observable phenotype whereas a knockout of GABA catabolic pathway genes led to growth and developmental defects under standard growth conditions. The implications of this study to our understanding of the action and function of GABA and GAD in crops are discussed.

Injectisome T3SS subunits as potential chaperones in the extracellular export of Pectobacterium carotovorum subsp. carotovorum bacteriocins Carocin S1 and Carocin S3 secreted via flagellar T3SS.

Pectobacterium carotovorum subsp. carotovorum (Pcc) causes soft-rot disease in a wide variety of plants resulting in economic losses worldwide. It produces various types of bacteriocin to compete against related plant pathogens. Studies on how bacteriocins are extracellularly secreted are conducted to understand the mechanism of interbacterial competition. In this study, the secretion of the low-molecular-weight bacteriocins (LMWB) Carocin S1 and Carocin S3 produced by a multiple-bacteriocin producing strain of Pcc, 89-H-4, was investigated. Tn5 insertional mutagenesis was used to generate a mutant, TH22-6, incapable of LMWBs secretion. Sequence and homology analyses of the gene disrupted by transposon Tn5 insertion revealed that the gene sctT, an essential component of the injectisome type III secretion machinery (T3aSS), is required for the secretion of the bacteriocins. This result raised a question regarding the nature of the secretion mechanism of Pcc bacteriocins which was previously discovered to be secreted via T3bSS, a system that utilizes the bacterial flagellum for extracellular secretions. Our previous report has shown that bacteriocin Carocin S1 cannot be secreted by mutants that are defective of T3bSS-related genes such as flhA, flhC, flhD and fliC. We knocked out several genes making up the significant structural components of both T3aSS and T3bSS. The findings led us to hypothesize the potential roles of the T3aSS-related proteins, SctT, SctU and SctV, as flagellar T3SS chaperones in the secretion of Pcc bacteriocins. This current discovery and the findings of our previous study helped us to conceptualize a unique Type III secretion system for bacteriocin extracellular export which is a hybrid of the injectisome and flagellar secretion systems.

The TOR kinase pathway is relevant for nitrogen signaling and antagonism of the mycoparasite Trichoderma atroviride.

Trichoderma atroviride (Ascomycota, Sordariomycetes) is a well-known mycoparasite applied for protecting plants against fungal pathogens. Its mycoparasitic activity involves processes shared with plant and human pathogenic fungi such as the production of cell wall degrading enzymes and secondary metabolites and is tightly regulated by environmental cues. In eukaryotes, the conserved Target of Rapamycin (TOR) kinase serves as a central regulator of cellular growth in response to nutrient availability. Here we describe how alteration of the activity of TOR1, the single and essential TOR kinase of T. atroviride, by treatment with chemical TOR inhibitors or by genetic manipulation of selected TOR pathway components affected various cellular functions. Loss of TSC1 and TSC2, that are negative regulators of TOR complex 1 (TORC1) in mammalian cells, resulted in altered nitrogen source-dependent growth of T. atroviride, reduced mycoparasitic overgrowth and, in the case of Δtsc1, a diminished production of numerous secondary metabolites. Deletion of the gene encoding the GTPase RHE2, whose mammalian orthologue activates mTORC1, led to rapamycin hypersensitivity and altered secondary metabolism, but had an only minor effect on vegetative growth and mycoparasitic overgrowth. The latter also applied to mutants missing the npr1-1 gene that encodes a fungus-specific kinase known as TOR target in yeast. Genome-wide transcriptome analysis confirmed TOR1 as a regulatory hub that governs T. atroviride metabolism and processes associated to ribosome biogenesis, gene expression and translation. In addition, mycoparasitism-relevant genes encoding terpenoid and polyketide synthases, peptidases, glycoside hydrolases, small secreted cysteine-rich proteins, and G protein coupled receptors emerged as TOR1 targets. Our results provide the first in-depth insights into TOR signaling in a fungal mycoparasite and emphasize its importance in the regulation of processes that critically contribute to the antagonistic activity of T. atroviride.

The Membrane-Anchoring Region of the AcMNPV P74 Protein Is Expendable or Interchangeable with Homologs from Other Species.

Baculoviruses are insect pathogens that are characterized by assembling the viral dsDNA into two different enveloped virions during an infective cycle: occluded virions (ODVs; immersed in a protein matrix known as occlusion body) and budded virions (BVs). ODVs are responsible for the primary infection in midgut cells of susceptible larvae thanks to the per os infectivity factor (PIF) complex, composed of at least nine essential viral proteins. Among them, P74 is a crucial factor whose activity has been identified as virus-specific. In this work, the p74 gene from AcMNPV was pseudogenized using CRISPR/Cas9 technology and then complemented with wild-type alleles from SeMNPV and HearSNPV species, as well as chimeras combining the P74 amino and carboxyl domains. The results on Spodoptera exigua and Rachiplusia nu larvae showed that an amino terminal sector of P74 (lacking two potential transmembrane regions but possessing a putative nuclear export signal) is sufficient to restore the virus infectivity whether alone or fused to the P74 transmembrane regions of the other evaluated viral species. These results provide novel information about the functional role of P74 and delimit the region on which mutagenesis could be applied to enhance viral activity and, thus, produce better biopesticides.