Tubulin Gene Research Articles

OsTUB1 confers salt insensitivity by interacting with Kinesin13A to stabilize microtubules and ion transporters in rice.

Salt stress is one of the major environmental factors limiting plant growth and development. Although microtubule (MT) organization is known to be involved in response to salt stress, few tubulin genes have been identified that confer salt insensitivity in plants. In this study, we identified a MT encoding gene, OsTUB1, that increased the survival rate of rice plants under salt stress by stabilizing MT organization and ion transporters. We found that OsTUB1 interacted with Kinesin13A protein, which was essential for OsTUB1-regulated MT organization under salt stress. Further molecular evidence revealed that a OsTUB1-Kinesin13A complex protected rice from salt stress by sustaining membrane-localized Na+ transporter OsHKT1;5, a key regulator of ionic homeostasis. Our results shed light on the function of tubulin and kinesin in regulating MT organization and stabilizing Na+ transporters and Na+ flux at the plasma membrane in rice. The identification of the OsTUB1-Kinesin13A complex provides novel genes for salt insensitivity rice breeding in areas with high soil salinity.

Aspergillus niger causes black mould disease on Piarom dates, the most economically valuable export date cultivar in southern Iran

In 2021, we observed symptoms of black mould disease on dates cv. Piarom, the most economically valuable export date cultivar in Hormozgan province, southern Iran. This study aimed to evaluate the disease incidence, identify the aetiological agent, and fulfill the pathogenicity on Piarom dates in this area. Aspergillus isolates were permanently obtained from the symptomatic dates on PDA, which were identified as Aspergillus niger based on morphological characteristics, as well as partial sequence data from the internal transcribed spacer region (ITS1-5.8S-ITS2) and Beta Tubulin (benA) gene. A phylogeny inferred using individual sequence data from ITS and benA sequences placed our isolates together with the other A. niger strains in the GenBank. The pathogenicity assays using symptom-free Piarom dates revealed the association of A. niger with the disease. Reisolation from the inoculated fruits yielded the isolates of A. niger, proving Koch's postulates. This is the first report of A. niger causing black mould disease on Piarom dates in Iran.

First Report of Lasiodiplodia pseudotheobromae Causing Collar Rot of Peanut in Shandong Province, China.

In August 2019, a collar rot of peanut was observed in several fields in Qingdao, Shandong province, China. Disease survey was conducted in several peanut fields. Less than 5% plants exhibited various symptoms, including brown or black stem rot, pod rot, leaf chlorotic, wilted, and even dead. Symptomatic stems were cut into small pieces, surface disinfested with 70% ethanol for 1 min, 1% NaClO for 2 minutes, rinsed three times with sterile water, and dried on sterile filter papers. Pieces then were plated on potato dextrose agar (PDA) media and incubated at 25°C in darkness. Fungal cultures were initially white, then turned gray, and eventually turned black, and aerial hyphae were dense, fluffy. Conidia were ellipsoidal, initially hyaline, unicellular, 14.3 to 21.1 × 8.7 to 13.2 µm (n = 50), and mature conidia showed dark brown, with a central septum, and longitudinal stripes. Molecular identification was performed by sequencing ITS with ITS1/ITS4 (White et al., 1990) and beta tubulin gene with Bt2a/Bt2b (Glass and Donaldson, 1995) of a representative isolate ZHX9. ITS and beta tubulin regions (OK427342 and OK489788) of ZHX9 obtained 99.62 and 100% similar to L. pseudotheobromae (KF766193 and EU673111), respectively. Phylogenetic analysis was done using Neighbor-Joining (NJ) analysis based on those gene sequences. The microorganism we have isolated was identified as L. pseudotheobromae based on molecular analysis and morphological characteristics. For pathogenicity assay, twelve ten-days-old peanut (Zhonghua No.12) seedlings were each inoculated with one mycelial plug (8 mm in diameter) by placing the inoculum on the base of the stem. Twelve plants were each inoculated with a plug of non-colonized PDA as controls. Plants were incubated in a growth chamber (30°C in the day and 25°C at night, a 12-h photoperiod and 80% RH). Necrotic lesions were observed on stems of all inoculated seedlings 5 days after inoculation, whereas control plants remained asymptomatic, and L. peudotheobromae was consistently re-isolated from symptomatic stem. In Asia, peanut collar rot caused by L. teudotheobromae has been reported in India, Indonesia, North Vietnam (Nguyen, et al., 2006) and China (Guo, et al., 2014), but collar rot caused by L. pseudotheobromae has not been reported. To our knowledge, this is the first report of L. peudotheobromae causing collar rot on peanut in China. These results will provide crucial information for studying on epidemiology and management of this disease.

Omics Analyses of Trichomonas vaginalis Actin and Tubulin and Their Participation in Intercellular Interactions and Cytokinesis.

Actin and tubulin proteins from Trichomonas vaginalis are crucial for morphogenesis and mitosis. This parasite has 10 and 11 genes coding bonafide actin and tubulin proteins, respectively. Hence, the goal of this work was to analyze these actin and tubulin genes, their expression at the mRNA and protein levels, and their parasite localization in intercellular interaction and cytokinesis. Representative bonafide actin (tvact1) and tubulin (tvtubα1) genes were cloned into and expressed in Escherichia coli. The recombinant proteins TvACT1r and TvTUBα1r were affinity purified and used as antigens to produce polyclonal antibodies. These antibodies were used in 1DE and 2DE WB and indirect immunofluorescence assays (IFA). By IFA, actin was detected as a ring on the periphery of ameboid, ovoid, and cold-induced cyst-like parasites, on pseudopods of amoeboid parasites, and in cytoplasmic extensions (filopodia) in cell–cell interactions. Tubulin was detected in the axostyle, flagellum, undulating membrane, and paradesmose during mitosis. Paradesmose was observed by IFA mainly during cytokinesis. By scanning electron microscopy, a tubulin-containing nanotubular structure similar to the tunneling nanotubes (TNTs) was also detected in the last stage of cytokinesis. In conclusion, actin and tubulin are multigene families differentially expressed that play important roles in intercellular interactions and cytokinesis.

New records of mycobiota associated with stored wheat and its by-products in Iraq

Abstract. Fadhil WF, Al-Saadoon AH, Al-Moussawi FM. 2022. New records of mycobiota associated with stored wheat and its by-products in Iraq. Biodiversitas 23: 3099-3107. Wheat is a staple food for the Iraqi population and is an important feed source due to its high nutritional value. Consequently, wheat and wheat by-products are susceptible to various species of fungi during processing, transportation, or storage. In this paper, thirty-two selected fungal isolates were subjected to morphological and molecular analysis for identification through combined sequences of ITS regions, actin, calmodulin, glyceraldehyde-3-phosphate dehydrogenase, and ?-tubulin genes according to the target fungal genus. The results showed that eighteen fungal species were identified from wheat grains, flour, and bran samples collected from different silos and mills in three provinces in southern Iraq. In addition, nine species were recorded for the first time from Iraq, i.e., Alternaria consortialis, A. japonica, A. lolii, A. multiformis, A. ventricosa, Aspergillus montevidensis, Cladosporium halotolerans, C. versiforme, and Staganosporopsis tanaceti. Brief descriptions of the new records are presented. This study represents an important addition to the mycobiota of Iraq.

Natural occurrence of entomopathogenic fungus, <i>Aschersonia aleyrodis</i> on citrus whitefly, <i>Dialeurodes citri</i> (Ashmead) in Kinnow mandarin in Punjab, India

Surveys were conducted during 2017 and 2018 in the citrus orchards of Punjab, India to record the incidence of different insect pests and their natural enemies. During October-December, Entomopathogenic Fungus (EPF), Aschersonia aleyrodis was found to infect nymphs and pupae of citrus whitefly, Dialeurodes citri on the lower leaf surface of Kinnow from the orchards of Hoshiarpur, Ludhiana, Mansa and Fazilka districts.The fungus was isolated from the infected nymphs and pupae and morphological studies were conducted to confirm the identity of the entomopathogenic fungus. Aschersonia aleyrodis was reported for the first time on D. citri under Punjab conditions and this EPF also confirmed by amplification and sequencing of beta tubulin gene showed 99.40 per cent identity in NCBI, GenBank. Hence further studies on the host range, interaction with other insect pests and parasitoids, survival and longevity should be conducted to explore the potential of this fungus as microbial biocontrol agent for citrus whitefly.

Cataloging targetable dependencies of head and neck cancer cell lines in the DepMap CRISPR screens.

6056 Background: The DepMap genome-wide loss of function CRISPR screens offer new insight into gene dependencies in HPV(-) head and neck squamous cell carcinoma (HNSCC) cell lines. We aimed to leverage this data to guide preclinical studies by cataloging targetable dependencies and identifying ones predicted to offer a therapeutic window. We also aimed to identify targets representing potential synthetic lethalities by testing for associations between genetic alterations and dependency profile. Methods: DepMap was queried for gene probability and effect scores in cell lines from 77 tumors, including 62 HPV(-) HNSCCs plus 15 ESCCs, which have comparable etiology, genetic features, and tissue of origin. A probability score of ≥ 0.5 was used as the threshold for essentiality. Essential genes were selected for analysis by 3 criteria: (1) presence in ≥10% cell lines, (2) lack of dependency in CRISPR screens of normal human cell lineages, and (3) designation as druggable by the Drug-Gene Interaction Database. Gene set enrichment analysis was performed using the Hallmark Gene Sets. DepMap gene effect scores were used to prioritize targets likely to have a useful therapeutic window based on median scores greater than for EGFR (0.676), a target with established albeit modest utility for HNSCC. The Open Targets platform was used to identify targets with inhibitors used in trials for other cancers and/or nonmalignant diseases. Associations between dependencies and genetic alterations were defined using two-sample t-tests, with filter conditions of p < 0.05 and effect size ≥1. Results: The 231 genes meeting selection criteria had a median gene effect score of 0.56. The criteria captured targets of standard therapeutic agents including TYMS (5-FU), tubulin genes (paclitaxel), EGFR (cetuximab), plus known oncogenes like PIK3CA. GSEA showed enrichment of known oncogenic signaling pathways including PI3K/AKT and JAK/STAT, as well as hallmark cancer processes like DNA repair and apoptosis. 90% were not known oncogenes cataloged in the OncoKB Database. 45 genes had a median gene effect score between that of EGFR and the median for common essential genes, including 7 without known cancer-promoting roles: OTOP1, DHRSX, UTP11, MBTPS1, SLC25A3, PPIAL4G, and RBM10. 17% had inhibitors that reached a non-HNSCC phase II trial, including 10 targets not previously tested in cancer. Novel associations between dependencies and genetic alterations included DDX3X with NOTCH1mut, ITGB1 with CDKN2Amut, and ATP1A1 with HRASmut. Conclusions: We catalog targetable dependencies in cell line models of HNSCC. While well-studied targets were captured, many genes lacked known roles in malignancy. Targets of inhibitors tested in other diseases provide new tools to guide preclinical studies. Association of some dependencies with known molecular subgroups in HNSCC may enhance use of cell line models to personalize therapy.

Regulation of Tubulin Gene Expression: From Isotype Identity to Functional Specialization.

Genomes of higher eukaryotes encode a large tubulin gene superfamily consisting of at least six α and six β-tubulin isotypes. While some α and β-tubulin isotypes are ubiquitously expressed, others are cell-type specific. The subset of α and β-tubulins that is expressed in a given cell type is defined transcriptionally. But the precise mechanisms of how cells choose which α and β isotypes to express and at what level remain poorly understood. Differential expression of tubulin isotypes is particularly prominent during development and in specialized cells, suggesting that some isotypes are better suited for certain cell type-specific functions. Recent studies begin to rationalize this phenomenon, uncovering important differences in tubulin isotype behavior and their impact on the biomechanical properties of the microtubule cytoskeleton. I summarize our understanding of the regulation of tubulin isotype expression, focusing on the role of these complex regulatory pathways in building a customized microtubule network best suited for cellular needs.

Assessment of RNA extraction protocols from cladocerans.

The usage of cladocerans as non-model organisms in ecotoxicological and risk assessment studies has intensified in recent years due to their ecological importance in aquatic ecosystems. The molecular assessment such as gene expression analysis has been introduced in ecotoxicological and risk assessment to link the expression of specific genes to a biological process in the cladocerans. The validity and accuracy of gene expression analysis depends on the quantity, quality and integrity of extracted ribonucleic acid (RNA) of the sample. However, the standard methods of RNA extraction from the cladocerans are still lacking. This study evaluates the extraction of RNA from tropical freshwater cladocerans Moina micrura using two methods: the phenol-chloroform extraction method (QIAzol) and a column-based kit (Qiagen Micro Kit). Glycogen was introduced in both approaches to enhance the recovery of extracted RNA and the extracted RNA was characterised using spectrophotometric analysis (NanoDrop), capillary electrophoresis (Bioanalyzer). Then, the extracted RNA was analysed with reverse transcription polymerase chain reaction (RT-PCR) to validate the RNA extraction method towards downstream gene expression analysis. The results indicate that the column-based kit is most suitable for the extraction of RNA from M. micrura, with the quantity (RNA concentration = 26.90 ± 6.89 ng/μl), quality (A260:230 = 1.95 ± 0.15, A280:230 = 1.85 ± 0.09) and integrity (RNA integrity number, RIN = 7.20 ± 0.16). The RT-PCR analysis shows that the method successfully amplified both alpha tubulin and actin gene at 33-35 cycles (i.e. Ct = 32.64 to 33.48). The results demonstrate that the addition of glycogen is only suitable for the phenol-chloroform extraction method. RNA extraction with high and comprehensive quality control assessment will increase the accuracy and reliability of downstream gene expression, thus providing more ecotoxicological data at the molecular biological level on other freshwater zooplankton species.

A longitudinal two-year survey of the prevalence of trypanosomes in domestic cattle in Ghana by massively parallel sequencing of barcoded amplicons.

BackgroundAnimal African Trypanosomiasis (AAT) is one of the most economically important diseases affecting livestock productivity in sub-Saharan Africa. The disease is caused by a broad range of Trypanosoma spp., infecting both wild and domesticated animals through cyclical and mechanical transmission. This study aimed to characterize trypanosomes present in cattle at regular intervals over two years in an AAT endemic and a non-endemic region of Ghana.Methodology/Principal findingsGroups of cattle at Accra and Adidome were selected based on their geographical location, tsetse fly density, prevalence of trypanosomiasis and the breed of cattle available. Blood for DNA extraction was collected at approximately four to five-week intervals over a two-year period. Trypanosome DNA were detected by a sensitive nested PCR targeting the tubulin gene array and massively parallel sequencing of barcoded amplicons. Analysis of the data was a semi-quantitative estimation of infection levels using read counts obtained from the sequencing as a proxy for infection levels. Majority of the cattle were infected with multiple species most of the time [190/259 (73%) at Adidome and 191/324 (59%) at Accra], with T. vivax being the most abundant. The level of infection and in particular T. vivax, was higher in Adidome, the location with a high density of tsetse flies. The infection level varied over the time course, the timings of this variation were not consistent and in Adidome it appeared to be independent of prophylactic treatment for trypanosome infection. Effect of gender or breed on infection levels was insignificant.Conclusions/SignificanceMost cattle were infected with low levels of several trypanosome species at both study sites, with T. vivax being the most abundant. The measurements of infection over time provided insight to the importance of the approach in identifying cattle that could suppress trypanosome infection over an extended time and may serve as reservoir.

Targetable Vulnerabilities of Head and Neck Cancer Cell Lines Detected by the DepMap CRISPR Screens

<h3>Purpose/Objective(s)</h3> The DepMap genome-wide loss of function CRISPR screens offer new insight into gene dependencies in HPV(-) head and neck squamous cell carcinoma (HNSCC) cell lines. We aimed to leverage this data to guide preclinical studies by cataloging targetable dependencies that are predicted to offer a therapeutic window. We also aimed to identify targets representing potential synthetic lethalities by testing for associations between genetic alterations and dependency profile. <h3>Materials/Methods</h3> DepMap was queried for gene probability and effect scores in cell lines from 77 tumors, including 62 HPV(-) HNSCCs plus 15 ESCCs, which have comparable etiology and genetic profile. A probability score of ≥ 0.5 was used as the threshold for essentiality. Essential genes were selected for analysis by 3 criteria: (1) presence in ≥10% cell lines, (2) lack of common essentiality in prior CRISPR screens of normal human cell lineages, and (3) designation as druggable by the Drug-Gene Interaction Database. Gene set enrichment analysis (GSEA) was performed using the Hallmark Gene Sets. DepMap gene effect scores were used to prioritize targets likely to have a useful therapeutic window based on median scores greater than for <i>EGFR</i> (0.66) but less than for common essential genes (1.0). The Open Targets platform was used to identify targets with inhibitors used in trials for other cancers and/or nonmalignant diseases. Associations between dependencies and genetic alterations were defined using two-sample t-tests, with filter conditions of p<0.05 and effect size ≥1. <h3>Results</h3> The 231 genes meeting selection criteria had a median gene effect score of 0.56. The criteria captured targets of standard therapeutic agents including <i>TYMS</i> (5-FU), tubulin genes (paclitaxel), <i>EGFR</i> (cetuximab), plus known oncogenes like <i>PIK3CA</i>. GSEA showed enrichment of known oncogenic signaling pathways including PI3K/AKT and JAK/STAT, as well as hallmark cancer processes like DNA repair and apoptosis. 90% were not known oncogenes in the OncoKB Database. 44 genes had a median gene effect score between that of <i>EGFR</i> and the median for common essential genes, including 7 without known cancer-promoting roles: <i>OTOP1, DHRSX, UTP11,MBTPS1, SLC25A3, PPIAL4G</i>, and <i>RBM10</i>. 17% had inhibitors that reached a non-HNSCC phase II trial, including 10 targets not previously tested in cancer. Novel associations between dependencies and genetic alterations included <i>DDX3X</i> with <i>NOTCH1mut, ITGB1</i> with <i>CDKN2Amut</i>, and <i>ATP1A1</i> with <i>HRASmut.</i> <h3>Conclusion</h3> We catalog numerous targetable dependencies in cell line models of HNSCC. While well-studied targets were captured, many genes lacked known roles in malignancy. Targets of inhibitors tested in other diseases provide further resources to guide preclinical studies. Association of some dependencies with known molecular subgroups in HNSCC may enhance use of cell line models to personalize therapy. <h3>Author Disclosure</h3> <b>A.C. Cao</b>: None. <b>P. Rajagopalan</b>: None. <b>P.A. Gimotty</b>: None. <b>R. Brody</b>: None. <b>D. Basu</b>: None.

A dominant tubulin mutation causes cerebellar neurodegeneration in a genetic model of tubulinopathy.

Mutations in tubulins cause distinct neurodevelopmental and degenerative diseases termed “tubulinopathies”; however, little is known about the functional requirements of tubulins or how mutations cause cell-specific pathologies. Here, we identify a mutation in the gene Tubb4a that causes degeneration of cerebellar granule neurons and myelination defects. We show that the neural phenotypes result from a cell type–specific enrichment of a dominant mutant form of Tubb4a relative to the expression other β-tubulin isotypes. Loss of Tubb4a function does not underlie cellular pathology but is compensated by the transcriptional up-regulation of related tubulin genes in a cell type–specific manner. This work establishes that the expression of a primary tubulin mutation in mature neurons is sufficient to promote cell-autonomous cell death, consistent with a causative association of microtubule dysfunction with neurodegenerative diseases. These studies provide evidence that mutations in tubulins cause specific phenotypes based on expression ratios of tubulin isotype genes.

Substantive Morphological Descriptions, Phylogenetic Analysis and Single Nucleotide Polymorphisms of Aspergillus Species From Foeniculum vulgare.

Ascomycetous fungi are found associated with a wide variety of substrates which range from fresh water to marine ecosystems, tropical to temperate forest soils and deserts, throughout the world over. These demystifying fungi exist as endophytes, pathogens and saprobes. They have been studied due to their ability to contaminate foods and feedstuffs, causing an elaboration of mycotoxins. The objectives of the study included extensive analyses of the morphological features of fungi, especially Aspergilli, which have been presented while studying them on specific mycological media. It is also an elaborate compilation of substantive macro- and micro-morphological characterization of different Aspergilli isolated from the spice Foeniculum vulgare used in India and other countries in the world. Further, a first of its kind attempt has been made to study their relative abundance and frequency of occurrence, molecular phylogeny and genetic relatedness to characterize the Aspergilli into specific sections, groups and clades. Single nucleotide polymorphism (SNP) analysis was carried out to evaluate the functional consequences of nucleotide variations, synonymous and non-synonymous mutations in the protein structure. The study resulted in a total of 3,506 Aspergillus isolates, which were obtained from seventy (70) fennel samples, representing 14 Aspergillus species. The two most frequently found species were A. niger and A. flavus with a relative abundance of 32.24 and 11.63%, respectively. The taxonomy and current placements have been reappraised with suggestions and prospects for future research from six sections namely Terrei, Flavi, Fumigati, Nidulantes, Nigri, and Versicolores. In addition, a total number of 27 isolates were studied and deposited at the National Centre for Biotechnology Information (NCBI) and five Aspergillus species have been identified and are being reported for the first time from the fennel seeds, based on partial sequence analysis of the official fungal barcode namely, Internal Transcribed Spacer (ITS) and a functional gene, beta tubulin gene locus, coupled with phenotypic characterization. SNPs for specific DNA regions have been used to identify variants in Aspergilli obtained from Indian fennel seeds for the first time. The need for a polyphasic approach of morphological identification and genetic characterization of Aspergilli from Foeniculum vulgare is addressed and presented here in adequate detail. Our current work makes extensive use of partial beta-tubulin gene sequences analyses to evaluate the association between SNPs in five Aspergillus species sections.

New report of Biscogniauxia rosacearum as a pathogen on almond trees in Iran

Biscogniauxia species are known as fungal trunk pathogens on various tree species in the world. During a survey of trunk diseases of fruit trees conducted in Iran, a branch dieback was observed on almond trees in Kerman province (in the Southeast of Iran). Evaluation of symptomatic branches revealed wood discoloration in cross-sections and the presence of fruiting bodies of an ascomycete fungus on their bark. A total of 31 fungal isolates were obtained, 13 isolates recovered from necrotic wood tissues and 18 isolates from fruiting bodies. These isolates were subjected to morphological analysis as well as sequencing analysis of the partial ITS-rDNA and beta tubulin gene sequences. These fungal isolates were identified as Biscogniauxia rosacearum. Results of the pathogenicity tests showed that this species is pathogenic on almond shoots. Based on our knowledge, this is the first report of this species on almond trees in Iran and in the world.

Suppression of Cortical Microtubule Reorientation and Stimulation of Cell Elongation in Arabidopsis Hypocotyls under Microgravity Conditions in Space.

How microgravity in space influences plant cell growth is an important issue for plant cell biology as well as space biology. We investigated the role of cortical microtubules in the stimulation of elongation growth in Arabidopsis (Arabidopsis thaliana) hypocotyls under microgravity conditions with the Resist Tubule space experiment. The epidermal cells in the lower half of the hypocotyls of wild-type Columbia were longer in microgravity than at on-orbit 1 g, which precipitated an increase in the entire hypocotyl length. In the apical region, cortical microtubules adjacent to the outer tangential wall were predominantly transverse to the long axis of the cell, whereas longitudinal microtubules were predominant in the basal region. In the 9th to 12th epidermal cells (1 to 3 mm) from the tip, where the modification of microtubule orientation from transverse to longitudinal directions (reorientation) occurred, cells with transverse microtubules increased, whereas those with longitudinal microtubules decreased in microgravity, and the average angle with respect to the transverse cell axis decreased, indicating that the reorientation was suppressed in microgravity. The expression of tubulin genes was suppressed in microgravity. These results suggest that under microgravity conditions, the expression of genes related to microtubule formation was downregulated, which may cause the suppression of microtubule reorientation from transverse to longitudinal directions, thereby stimulating cell elongation in Arabidopsis hypocotyls.

The quiet evolutionary response to cellular challenges.

The quiet evolutionary response to cellular challenges.

Mutations in the most divergent α‐tubulin isotype, α8‐tubulin, cause defective platelet biogenesis

BackgroundIn the panel of genes commonly associated with inherited macrothrombocytopenia, an important fraction encodes key cytoskeletal proteins such as tubulin isotypes, the building blocks of microtubules. Macrothrombocytopenia‐causing mutations have been identified in the TUBB1 and TUBA4A genes, emphasizing their importance in the formation of platelets and their marginal band, a unique microtubule ring‐like structure that supports the platelet typical disc‐shaped morphology. This raised the hypothesis that other tubulin isotypes normally expressed in platelets could play a similar role in their formation. ObjectivesTo assess whether tubulin isotype genes other than TUBA4A and TUBB1 could be implicated in inherited macrothrombocytopenia. MethodsWe used high throughput sequencing to screen a cohort of 448 French blood donors with mild thrombocytopenia for mutations in a panel of selected genes known or suspected to be involved in platelet biogenesis. ResultsWe identified six distinct novel mutations in TUBA8, which encodes the most‐divergent α‐tubulin, as the causative determinant of macrothrombocytopenia and platelet marginal band defects. Functionally, all TUBA8 mutations were found to fully or partially inhibit the incorporation of the mutated α8‐tubulin in the microtubule network. ConclusionThis study provides strong support for a key role of multiple tubulin genes in platelet biogenesis by discovering variants in a tubulin gene that was previously not known to be important for platelets.

Research Progress on the Mechanism of Tubulin in Megakaryopoiesis and Regulation of Platelet Count--Review

Tubulin affects platelets count through the control of mitosis and the formation of pro-platelets during the maturation of megakaryoblast to platelets. Tubulin is involved in maintaining the integrity of platelet skeleton, and also participates in the change of platelet morphology during platelet activation. Some new anti-tumor drugs targeting cell mitosis are trying to reduce the effect on tubulin in order to reduce the side effect of drugs on platelet formation. In some patients with thrombocytopenia, the variation and polymorphism of the tubulin gene affect the structure of microtubule multimers, which leads to the decrease of platelet formation. This review summarized the latest progresses of tubulin in the regulation of megakaryopoiesis and thrombopoiesis.

First Report of Gray Leaf Spot Caused by Pyricularia oryzae (synonym: Magnaporthe oryzae) in Oat (Avena sativa) in Georgia, USA.

In southeastern U.S., oat (Avena sativa L.) is predominantly grown as a grain or forage crop due to its exceptional palatability (Buntin et al. 2009). In November 2020, leaf spot symptoms were observed in an oat field (cv. Horizon 720) in Screven County, Georgia (GPS: 32°38'57.6"N 81°31'32.178"W). Lesions were oblong, whitish to gray in color, and surrounded by dark brown borders. Symptomatic oat leaves were sampled from the field and cut into 1 cm2 sections that were surface sterilized, plated onto Potato Dextrose Agar (PDA) media and incubated in the dark at 23°C. To obtain pure cultures, fungal hyphal tips were transferred onto fresh PDA plates 3 times. The pathogen was identified as Pyricularia (Magnaporthe) based on typical conidial morphology (Ellis 1971). Conidia were hyaline, pyriform, 2-septate, and displayed a basal hilum. Conidia measured 5.32 to 10.64 μm (average 8.24 μm) wide by 15.96 to 29.26 μm (average 25.40 μm) long. The identification of Pyricularia was further confirmed genetically via PCR amplification followed by sequencing. Genomic DNA was extracted from a 14-day old pure culture using a CTAB method (Doyle and Doyle 1987). The internal transcribed spacer (ITS) region of ribosomal DNA, calmodulin (CaM) gene, and -tubulin (TUB) gene were amplified using ITS5-ITS4 (White et al. 1990), CMD5-CMD6 (Hong et al. 2005), and Bt2a- Bt2b (Glass and Donaldson 1995) primer sets, respectively. Amplicons were Sanger sequenced and blasted against the NCBI database. Results exhibited 100% (ITS), 100% (CaM), and 99.61% (TUB) homology with Pyricularia oryzae Cavara (GenBank accession no. LC554423.1, CP050920.1, and CP050924.1, respectively). The ITS, CaM, and TUB sequences of the isolate were deposited in GenBank as MZ295207, MZ342893, and MZ342894, respectively. In a greenhouse (23°C, 80% RH), Koch's postulates were carried out by using oat seedlings cv. Horizon 270 grown in Kord sheet pots filled with Sun Gro professional growing mix, and a P. oryzae spore suspension containing 104 conidia ml-1. The spore suspension (10 ml) was sprayed with an air sprayer onto 7 pots of oat seedlings at the two-leaf stage. Seven supplementary pots of oat seedlings of the same cultivar were sprayed with sterile water to act as controls. After inoculation, plants were covered with black plastic bags that had been sprayed with sterile water to maintain high humidity and incubated overnight in the greenhouse. The bags were removed the next day, and plants were evaluated for symptoms in the following days. Seven days after inoculation, plants displayed symptoms similar to those found in the original field sample. Control plants showed no symptoms. Pyricularia oryzae was consistently re-isolated from inoculated symptomatic oat tissues. To our knowledge, this is the first report of gray leaf spot caused by P. oryzae on oat in the state of Georgia and in the continental United States. Pyricularia oryzae can infect several graminaceous plants, including agronomically important crops such as rice (Oryza sativa) and wheat (Triticum spp.) (Chung et al. 2020). Phylogenetic analysis on the ITS region using 6 different host lineages was performed and revealed that this oat isolate was most closely related to the Lolium lineage. This outbreak could have economic implications in oat production.

Polyphasic identification of decay agents of lemon fruits in Serbia

Lemon fruits are an important source of vitamin C, potassium, folate, carotenoids, polyphenols, coumarins and terpenes. These lemon compounds have antioxidant and anti-inflammatory properties which have beneficial effects on human health. This research aimed to elucidate the etiology of blue and green molds detected on lemon fruits in Serbia. Using integrative identification approach, the obtained isolates were characterized from morphological, physiological, molecular, phylogenetic and pathological aspects. Colony growth and morphology were examined on Czapek yeast autolysate agar (CYA), Malt extract agar (MEA) and Creatine sucrose agar (CREA), and on CYA at two additional incubation temperatures (5 and 37 ?C). For molecular identification, ITS and partial ?-tubulin (BenA) genes were sequenced. Phylogenetic relationships were investigated using maximum-likelihood method. A pathogenicity test was carried out and the possible difference in pathogenicity among isolates was assessed with analysis of variance (ANOVA) and subsequent Tukey?s test. Four species were identified: Penicillium expansum, Penicillium digitatum, Penicillium polonicum and Talaromyces rugulosus. All four species proved to be pathogenic on lemon fruits, producing symptoms similar to those observed on naturally infected fruits. The results of this study are the first records of the beforementioned Penicillium/ Talaromyces species as postharvest pathogens on lemon fruits in Serbia and the first world report of T. rugulosus as phytopathogenic on the same host.