Kinetically Stabilizing Mutations in Beta Tubulins Create Isotype-Specific Brain Malformations

Mutations in the family of genes encoding the tubulin subunits of microtubules are associated with a spectrum of human brain malformations known as tubulinopathies. How these mutations impact tubulin activity to give rise to distinct developmental consequences is poorly understood. Here we report two patients exhibiting brain malformations characteristic of tubulinopathies and heterozygous T178M missense mutations in different β-tubulin genes, TUBB2A or TUBB3. RNAseq analysis indicates that both TUBB2A and TUBB3 are expressed in the brain during development, but only TUBB2A maintains high expression in neurons into adulthood. The T178 residue is highly conserved in β-tubulins and located in the exchangeable GTP-binding pocket of β-tubulin. To determine the impact of T178M on β-tubulin function we created an analogous mutation in the β-tubulin of budding yeast and show that the substitution acts dominantly to produce kinetically stabilized microtubules that assemble and disassemble slowly, with fewer transitions between these states. In vitro experiments with purified mutant tubulin demonstrate that T178M decreases the intrinsic assembly activity of β-tubulin and forms microtubules that rarely transition to disassembly. We provide evidence that the T178M substitution disrupts GTPase-dependent conformational changes in tubulin, providing a mechanistic explanation for kinetic stabilization. Our findings demonstrate the importance of tubulin’s GTPase activity during brain development, and indicate that tubulin isotypes play different, important roles during brain development.

Characterization of the β-tubulin gene family in Ascaris lumbricoides and Ascaris suum and its implication for the molecular detection of benzimidazole resistance

Background The treatment coverage of control programs providing benzimidazole (BZ) drugs to eliminate the morbidity caused by soil-transmitted helminths (STHs) is unprecedently high. This high drug pressure may result in the development of BZ resistance in STHs and so there is an urgent need for surveillance systems detecting molecular markers associated with BZ resistance. A critical prerequisite to develop such systems is an understanding of the gene family encoding β-tubulin proteins, the principal targets of BZ drugs. Methodology and principal findings First, the β-tubulin gene families of Ascaris lumbricoides and Ascaris suum were characterized through the analysis of published genomes. Second, RNA-seq and RT-PCR analyses on cDNA were applied to determine the transcription profiles of the different gene family members. The results revealed that Ascaris species have at least seven different β-tubulin genes of which two are highly expressed during the entire lifecycle. Third, deep amplicon sequencing was performed on these two genes in more than 200 adult A. lumbricoides (Ethiopia and Tanzania) and A. suum (Belgium) worms, to investigate the intra- and inter-species genetic diversity and the presence of single nucleotide polymorphisms (SNPs) that are associated with BZ resistance in other helminth species; F167Y (TTC>TAC or TTT>TAT), E198A (GAA>GCA or GAG>GCG), E198L (GAA>TTA) and F200Y (TTC>TAC or TTT>TAT). These particular SNPs were absent in the two investigated genes in all three Ascaris populations. Significance This study demonstrated the presence of at least seven β-tubulin genes in Ascaris worms. A new nomenclature was proposed and prioritization of genes for future BZ resistance research was discussed. This is the first comprehensive description of the β-tubulin gene family in Ascaris and provides a framework to investigate the prevalence and potential role of β-tubulin sequence polymorphisms in BZ resistance in a more systematic manner than previously possible.

Genetic polymorphism of white mistletoe (Viscum album L.) in Ukraine

Aim. The aim of the study was to establish genetic differences between V. album growing in different parts of Ukraine. Methods. White mistletoe samples collected in different regions of Ukraine were used in the study. The method of estimating the intron length polymorphism of β-tubulin genes was used. Amplified DNA fragments were fractionated by non-denaturing polyacrylamide gel electrophoresis and visualized by silver nitrate staining. Results. The genotypes of 91 white mistletoe plants were analyzed. DNA profiles of white mistletoe with a specific amplicons of β-tubulin gene introns were obtained, which allowed to differentiate the samples from each other. Fingerprinting data were used for cluster analysis and dendrogram construction. Conclusions. It was found that the analyzed mistletoe samples did not differ by geographical factor and were characterized by a low level of genetic diversity in the studied samples. Keywords: Viscum album L., intron length polymorphism, β-tubulin, genetic variability, Ukraine.

Understanding Resistance Mechanisms to Trifluralin in an Arkansas Palmer Amaranth Population

Amaranthus palmeri S. Watson (Palmer amaranth) is considered a problematic and troublesome weed species in many crops in the USA, partly because of its ability to evolve resistance to herbicides. In this study, we explored the mechanism of resistance in a trifluralin-resistant A. palmeri accession collected from Arkansas, USA. Dose-response assays using agar plates demonstrated an EC50 (effective concentration that reduces root length by 50%) of 1.02 µM trifluralin compared to 0.39 µM obtained in the susceptible accession. Thus, under these conditions, the resistant accession required 2.6 times more trifluralin to inhibit root length by 50%. Seeds in the presence or absence of the cytochrome P450-inhibitior malathion displayed a differential response with no significant influence on root length, suggesting that resistance is not P450-mediated. In addition, application of 4-chloro-7-nitrobenzofurazan (NBD-Cl), a glutathione S-transferase (GST) inhibitor, showed significant differences in root length, indicating that GSTs are most likely involved in the resistance mechanism. Sequencing of α- and β-tubulin genes revealed no single nucleotide polymorphisms (SNPs) previously described between accessions. In addition, relative gene copy number of α- and β-tubulin genes were estimated; however, both resistant and susceptible accessions displayed similar gene copy numbers. Overall, our results revealed that GST-mediated metabolism contributes to trifluralin resistance in this A. palmeri accession from Arkansas.

First report of Phaeoacremonium oleae and P. viticola associated with olive trunk diseases in Italy

Over 300 trunk, branch and stem samples with vascular discolouration, necrotic wood and shoot death were collected from olive (Olea europaea) orchards in Lecce, Brindisi, Bari and Foggia provinces (Apulia region, Italy) from October to May from 2013 to 2019. Small chips of symptomatic wood samples were surface sterilised (5% NaOCl, 3 min; 70% ethanol, 30 s), rinsed (sterile distilled water, ×3), and placed onto potato dextrose agar (PDA) plates amended with 500 ppm streptomycin sulphate. After 14 days at 25 °C in the dark, hyphal tips of growing fungi, including different taxa, for instance Phaeoacremonium and Botryosphaeriaceae spp., were transferred to new PDA plates and incubated until sporulation. Monoclonal colonies resembling Phaeoacremonium-like genus (Mostert et al. 2006) were selected for further study, and genomic DNA of 59 representative isolates was extracted (Carlucci et al. 2013). Partial actin and β-tubulin genes were amplified with primers ACT-513F/ACT-783R (Carbone & Kohn 1999), and T1(O’Donnell & Cigelnik 1997) and Bt2b (Glass & Donaldson 1995), respectively. The sequenced amplicons were compared by BLAST algorithms with reference strains of Phaeoacremonium spp. retrieved from GenBank. Forty-four isolates showed 99% to 100% similarity with reference strains P. italicum, P. minimum, P. parasiticum, P. scolyti and P. sicilianum (Carlucci et al. 2015), nine with P. oleae, and six with P. viticola. Actin and β-tubulin sequences of P. oleae (Pm14) and P. viticola (Pm34) were submitted to GenBank (MW714561, MW714563; MZ318697, MZ318696). Microscopy of P. oleae isolates showed: conidiophores branched and unbranched, (18.7–)21.9–57.1(–67.8) × (2.9–)3.3–4.7(–5.2) (mean, 38.9×4.1) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.4 to 5.5 μm long, and 1.5 to 2.4 (mean, 4.6 × 2.2) μm wide (n=30). Microscopy of P. viticola isolates showed: conidiophores subcylindrical, branched at base (6.7–)8.9–27.2(–29.3) × (2.0–)2.6–3.3(–3.7) (mean, 21.4 × 3.2) μm (n=30); conidia oblong-ellipsoidal to obovoid or subcylindrical 3.3 to 6.8 μm long, and 1.1 to 2.2 (mean, 4.2 × 1.6) μm wide (n=30). In spring 2020, artificial inoculations were carried out with P. oleae (Pm14, Pm46) and P. viticola (Pm34, Pm43) strains on 10 healthy, 2-year-old olive seedlings cultivar ‘Coratina’. Agar plugs (diameter, 0.3–0.5 cm) from 10-day-old cultures grown on water agar at 23 (±2) °C were inserted under the bark of small wounds in the stems (length, 0.4–1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten control olive seedlings were inoculated with sterile agar plugs. The experiment was replicated three times. All inoculated young olive plants were grown in pots in a greenhouse without temperature control. After 120 days, inoculated plants showed decline symptoms, and when cut longitudinally, brown streaks were observed in the wood. For P. oleae these streaks measured 3.0-5.5 cm long (standard deviation [SD], 0.9 cm, and for P. viticola they were 1.8-3.5 cm (SD, 0.62). Both fungal species were re-isolated from the symptomatic wood from 85% and 80%, respectively, of these inoculated olive seedlings, fulfilling Koch’s postulates. No symptoms were observed from olive seedlings used as control. P. oleae was first described as a fungal pathogen of wild olive (Olea europaea subsp. cuspidate) in South Africa by Spies et al. (2018), and P. viticola as a fungal pathogen of grapevine in France by Dupont et al. (2000). To the best of our knowledge, this is the first report of P. oleae associated with olive trunk disease in Italy, and the first report of P. viticola associated with olive trunk disease worldwide. References: Carbone I. & Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2015. Eur. J. Plant Pathol. 141:717. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Dupont et al. 2000. Mycologia 92:499-504. Glass N. L. & Donaldson G. C. 1995. J. Cl. Microbiol. 41: 1332. Mostert L. et al. 2006. Stud. Mycol. 54:1. O’Donnell K. & Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103. Spies et al. 2018. Persoonia 40:26.

First report of Phaeoacremonium amygdalinum associated with almond dieback and wood disease in Italy

In the last 10 years, almond (Prunus dulcis (Mill.) Webb) cultivation areas in the region of Apulia (southern Italy) have increased. A recent survey in young almond orchards recorded characteristic disease symptoms including rapid collapse of branches, chlorosis of leaves (with sudden wilting and death) in June/July, and bud and shoot dieback in December-March. Internal wood symptoms on stems consisted of brown vascular streaking that led to death of young apical shoots within a few weeks. Wood samples were collected and taken to the laboratory, where they underwent disinfection according to Fisher et al. (2002). Fungal isolation techniques were applied, with small pieces of wood tissue (0.5-1.0 cm) placed on potato dextrose agar (PDA; Oxoid Ltd.) supplemented with 300 mg/L streptomycin sulphate, and incubated at 23 ±2 °C in the dark. After 10 days, based on morphological features carried out by microscopic studies, 85% (n = 55) of the resulting fungal colonies (N = 65) were preliminarily attributed to known fungal pathogens of woody tissues, of which 12.4% (n = 8) belonged to the Phaeoacremonium genus. These last were purified by transferring single germinating conidia onto fresh PDA plates. In particular, the conidiophores were mostly short, usually unbranched, erect to flexuous, up to 5-septate, brown to pale brown, verruculose on the lower part, (12–)15.7–41(–55) (av. -29) μm long and 1.6–3.2 (av. 2.1) μm wide (n = 30). Conidia hyaline, oblong ellipsoidal-obovoid or subcylindrical (3–)4–5 μm long and 1.5–3 (av. = 4.4 × 2.1) μm wide (n =30). All Phaeoacremonium isolates were successfully subjected to genomic DNA extraction, according to Carlucci et al. (2013). Partial actin and β-tubulin genes were amplified with the ACT-513F/ACT-783R (Carbone and Kohn 1999) and T1 (O’Donnell and Cigelnik 1997) and Bt2b (Glass and Donaldson 1995) primers, respectively. Consensus sequences were compared with reference strains (extype) in the GenBank database, using the Basic Local Alignment Search Tool (BLAST). In particular, all isolates showed 99-100% similarity with reference strains of Phaeoacremonium amygdalinum Gramaje, Armengol & L. Mostert (JN191301; JN191305). Actin and β-tubulin sequences of P. amygdalinum strain Pm10 were deposited in GenBank (MW716265; MW714562). In spring 2020, artificial inoculations with two isolates of P. amygdalinum (Pm10; Pm15) were carried out on 10 healthy, 2-year-old almond seedlings of cv. ‘Filippo Cea’. Agar plugs (diameter, 0.3–0.5 cm) taken from 10-day-old cultures grown on PDA at 23 ±2 °C were inserted into small wounds under the bark of the stems (length, 0.4–1.0 cm) made with a sterile scalpel. After inoculation, the wounds were wrapped with wet sterile cotton wool and sealed with Parafilm. Ten almond seedling were used as controls, being inoculated with sterile agar plugs. The experiment was replicated three times. The inoculated young almond plants were grown in pots in a greenhouse without temperature control. After 150 days, the inoculated plants showed decline symptoms and internal longitudinal brown wood streaking (average, length, 2.7–6.4 cm). Phaeoacremonium amygdalinum was reisolated from symptomatic wood of 95% of the experimental almond seedlings, thus fulfilling Koch’s postulates. No symptoms were observed on almond seedling used as controls. Phaeoacremonium amygdalinum was first described as a fungal pathogen of almond in Spain (Gramaje et al., 2012). To the best of our knowledge, this is the first report of P. amygdalinum associated with almond dieback disease in Italy. References Carbone I. and Kohn L.M. 1999. Mycologia 91:553. Carlucci A. et al. 2013. Phytopathol. Mediterr. 52:517. Fisher et al 2002. Mycol. Prog. 1:315 Glass N. L. and Donaldson G. C. 1995. J. Cl. Microbiol. 41:1332. Gramaje et al. 2012. Persoonia 28:1. O’Donnell K. and Cigelnik E. 1997. Mol. Phylogenetics Evol 7:103.

Genome-Wide Analysis of Tubulin Gene Family in Cassava and Expression of Family Member FtsZ2-1 during Various Stress

Filamentous temperature-sensitive protein Z (Tubulin/FtsZ) family is a group of conserved GTP-binding (guanine nucleotide-binding) proteins, which are closely related to plant tissue development and organ formation as the major component of the cytoskeleton. According to the published genome sequence information of cassava (Manihot esculenta Crantz), 23 tubulin genes (MeTubulins) were identified, which were divided into four main groups based on their type and phylogenetic characteristics. The same grouping generally has the same or similar motif composition and exon–intron structure. Collinear analysis showed that fragment repetition event is the main factor in amplification of cassava tubulin superfamily gene. The expression profiles of MeTubulin genes in various tissue were analyzed, and it was found that MeTubulins were mainly expressed in leaf, petiole, and stem, while FtsZ2-1 was highly expressed in storage root. The qRT-PCR results of the FtsZ2-1 gene under hormone and abiotic stresses showed that indole-3-acetic acid (IAA) and gibberellin A3 (GA3) stresses could significantly increase the expression of the FtsZ2-1 gene, thereby revealing the potential role of FtsZ2-1 in IAA and GA3 stress-induced responses.

Susceptible trichostrongyloid species mask presence of benzimidazole-resistant Haemonchus contortus in cattle

Background Benzimidazole (BZ) anthelmintics are widely used to control infections with parasitic nematodes, but BZ resistance is an emerging threat among several nematode species infecting humans and animals. In Sudan, BZ-resistant Haemonchus contortus populations were recently reported in goats in South Darfur State. The objective of this study was to collect data regarding the situation of BZ resistance in cattle parasitic nematodes in South Darfur using phenotypic and molecular approaches, besides providing some epidemiological data on nematodes in cattle. Methods The faecal egg count reduction test and the egg hatch test (EHT) were used to evaluate benzimidazole efficacy in cattle nematodes in five South Darfur study areas: Beleil, Kass, Nyala, Rehed Al-Birdi and Tulus. Genomic DNA was extracted from pools of third-stage larvae (L3) (n = 40) during trials, before and after treatment, and pools of adult male Haemonchus spp. (n = 18) from abattoirs. The polymorphisms F167Y, E198A and F200Y in isotype 1 β-tubulin genes of H. contortus and H. placei were analysed using Sanger and pyrosequencing. Results Prevalence of gastro-intestinal helminths in cattle was 71% (313/443). Reduced albendazole faecal egg count reduction efficacy was detected in three study areas: Nyala (93.7%), Rehed Al-Birdi (89.7%) and Tulus (88.2%). In the EHT, EC50 values of these study areas ranged between 0.032 and 0.037 µg/ml thiabendazole. Genus-specific PCRs detected the genera Haemonchus, Trichostrongylus and Cooperia in L3 samples collected after albendazole treatment. Sanger sequencing followed by pyrosequencing assays did not detect elevated frequencies of known BZ resistance-associated alleles in codon F167Y, E198A and F200Y in isotype 1 β-tubulin gene of H. placei (≤ 11.38%). However, polymorphisms were detected in H. contortus and in samples with mixed infections with H. contortus and H. placei at codon 198, including E198L (16/58), E198V (2/58) and potentially E198Stop (1/58). All pooled L3 samples post-albendazole treatment (n = 13) were identified as H. contortus with an E198L substitution at codon 198. Conclusions To the knowledge of the authors, this is the first report of reduced albendazole efficacy in cattle in Sudan and is the first study describing an E198L substitution in phenotypically BZ-resistant nematodes collected from cattle.

First report of Seimatosporium vitifusiforme causing trunk disease in Chilean grapevines (Vitis vinifera)

Grapevine is one of the most important fruit crops in Chile and trunk diseases reduce the productivity, quality, and longevity of the vineyards. A survey was conducted in ancient (> 50 years) vineyards of Cauquenes (35°57´14´´S 72°17´07´´W) and Itata valleys (36°38´13´´S 72°30´57´´W), located in the central area of Chile, during 2019. Trunks and cordons showing dieback and dark brown to black wood discoloration were collected from 50 to 200-year-old plants of six cultivars: País, Moscatel, Torontel Amarilla, Carignan, Aliatica and Aligote. The bark was removed and 0.5-cm sections were cut from the edges of necrotic wood lesions. Subsequently, pieces were surface disinfected using 10% v/v sodium hypochlorite bleach (4.9% chlorine), plated on acidified quarter-strength potato dextrose agar (APDA) (25% PDA, acidified with 0.1% v/v 85% lactic acid) and incubated at 25°C, for 14 to 28 days. Hyphal tips were excised and transferred to PDA to obtain pure cultures. Along with the conidiomata and conidia produced, growth rate, color and shape of the colonies on PDA, after 7 and 14 days of incubation at 25°C (n=17), were recorded. DNA was extracted from pure cultures of three isolates on PDA: HMV3, HMV64 and HMV81. The internal transcribed spacer region and partial β-tubulin genes were amplified, using ITS1/ITS4 (White et al. 1990) and bt2A/bt2B (Glass & Donaldson 1995) primers, respectively. Sequences were subjected to NCBI BLAST search and compared to the published sequences. Isolated colonies were whitish to light-brown, cottony with a smooth margin (n=37). Their mycelium grew 1.9 cm after 7-days and 3.2 cm after 14-days of incubation on PDA, at 25°C. Colonies produced black globose pycnidia and curved, slightly-pigmentated, three-septated conidia 22.3-(29.8)-32.2 x 3.9-(4.8)-5.3 µm (n=30), with apical and basal flexuous appendages 4.3-(12.7)-21.5 µm (n=20). When compared to type sequences of Seimatosporium vitifusiforme (Lawrence et al. 2018), ITS and βtub sequences identity of these isolates were 99 to 100% identical. To produce uniform healthy plants for pathogenicity tests, Petit Syrah canes (1-year old) were rooted in tap water amended with 500 ppm of indole-butyric acid, for 30 days. Plants were inoculated with 0.5-cm diameter mycelial plugs of actively growing colonies of the isolates HMV3, HMV64 and HMV81 (GenBank accessions no. MW026664, MW048518; MW026665, MW048519, and MW026666, MW048520, respectively). Sterile agar plugs were used for controls. Five plants per pathogen isolate were incubated at 25°C, in a humid chamber, for 25 days, and seven additional plants per isolate were incubated in aerated tap water, for 55 days. After the incubation period, the bark was removed and the lesions were measured. Dark necrotic lesions identical to the original observations were reproduced, both in the high humidity chamber (6% length) and water (10% length). There were no differences in lesion length among the isolates (P < 0.05). Control vines remained asymptomatic. To fulfill Koch´s postulates, isolations were made from symptomatic vines and compared to the ones used for inoculation, and found to be identical. Seimatosporium vitifusiforme was previously reported as a pathogen of Vitis vinifera in California, USA (Lawrence et al. 2018). Consequently, this is the second report of this fungus as a grapevine pathogen and the first one affecting Latin-American grapevines.