Large Multigene Family Research Articles

Genome-Wide Identification and Expression Analyses of the Thaumatin-Like Protein Gene Family in Tetragonia tetragonoides (Pall.) Kuntze Reveal Their Functions in Abiotic Stress Responses.

Thaumatin-like proteins (TLPs), including osmotins, are multifunctional proteins related to plant biotic and abiotic stress responses. TLPs are often present as large multigene families. Tetragonia tetragonoides (Pall.) Kuntze (Aizoaceae, 2n = 2x = 32), a vegetable used in both food and medicine, is a halophyte that is widely distributed in the coastal areas of the tropics and subtropics. Saline-alkaline soils and drought are two major abiotic stress factors significantly affecting the distribution of tropical coastal plants. The expression of stress resistance genes would help to alleviate the cellular damage caused by abiotic stress factors such as high temperature, salinity-alkalinity, and drought. This study aimed to better understand the functions of TLPs in the natural ecological adaptability of T. tetragonoides to harsh habitats. In the present study, we used bioinformatics approaches to identify 37 TtTLP genes as gene family members in the T. tetragonoides genome, with the purpose of understanding their roles in different developmental processes and the adaptation to harsh growth conditions in tropical coral regions. All of the TtTLPs were irregularly distributed across 32 chromosomes, and these gene family members were examined for conserved motifs of their coding proteins and gene structure. Expression analysis based on RNA sequencing and subsequent qRT-PCR showed that the transcripts of some TtTLPs were decreased or accumulated with tissue specificity, and under environmental stress challenges, multiple TtTLPs exhibited changeable expression patterns at short (2 h), long (48 h), or both stages. The expression pattern changes in TtTLPs provided a more comprehensive overview of this gene family being involved in multiple abiotic stress responses. Furthermore, several TtTLP genes were cloned and functionally identified using the yeast expression system. These findings not only increase our understanding of the role that TLPs play in mediating halophyte adaptation to extreme environments but also improve our knowledge of plant TLP evolution. This study also provides a basis and reference for future research on the roles of plant TLPs in stress tolerance and ecological environment suitability.

Unraveling Desmin's Head Domain Structure and Function.

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

The Identification, Characterization, and Functional Analysis of the Sugar Transporter Gene Family of the Rice False Smut Pathogen, Villosiclava virens.

False smut, caused by Villosiclava virens, is becoming increasingly serious in modern rice production systems, leading to yield losses and quality declines. Successful infection requires efficient acquisition of sucrose, abundant in rice panicles, as well as other sugars. Sugar transporters (STPs) may play an important role in this process. STPs belong to a major facilitator superfamily, which consists of large multigenic families necessary to partition sugars between fungal pathogens and their hosts. This study identified and characterized the STP family of V. viren, and further analyzed their gene functions to uncover their roles in interactions with rice. Through genome-wide and systematic bioinformatics analyses, 35 STPs were identified from V.virens and named from VvSTP1 to VvSTP35. Transmembrane domains, gene structures, and conserved motifs of VvSTPs have been identified and characterized through the bioinformatic analysis. In addition, a phylogenetic analysis revealed relationship between VvSTPs and STPs from the other three reference fungi. According to a qRT-PCR and RNA-sequencing analysis, VvSTP expression responded differently to different sole carbon sources and H2O2 treatments, and changed during the pathogenic process, suggesting that these proteins are involved in interactions with rice and potentially functional in pathogenesis. In total, 12 representative VvSTPs were knocked out through genetic recombination in order to analyze their roles in pathogenicity of V. virens. The knock-out mutants of VvSTPs showed little difference in mycelia growth and conidiation, indicating a single gene in this family cannot influence vegetative growth of V. virens. It is clear, however, that these mutants result in a change in infection efficiency in a different way, indicating that VvSTPs play an important role in the pathogenicity of virens. This study is expected to contribute to a better understanding of how host-derived sugars contribute to V. virens pathogenicity.

Olfactory receptor genes and chromosome 11 structural aberrations: Players or spectators?

The largest multi-gene family in metazoans is the family of olfactory receptor (OR) genes. Human ORs are organized in clusters over most chromosomes and seem to include >0.1% of the human genome. Because 369 out of 856 OR genes are mapped on chromosome 11 (HSA11), we sought whether they mediate structural rearrangements involving this chromosome. To this aim, we analyzed 220 specimens collected during diagnostic procedures involving structural rearrangements of chromosome 11. A total of 222 chromosomal abnormalities were included, consisting of inversions, deletions, translocations, duplications, and one insertion, detected by conventional chromosome analysis and/or FISH and array-CGH. We verified by bioinformatics and statistical approaches the occurrence of breakpoints in cytobands with or without OR genes. We found that OR genes are not involved in chromosome 11 reciprocal translocations, suggesting that different DNA motifs and mechanisms based on homology or non-homology recombination can cause chromosome 11 structural alterations. We also considered the proximity between the chromosomal territories of chromosome 11 and its partner chromosomes involved in the translocations by using the deposited Hi-C data concerning the possible occurrence of chromosome interactions. Interestingly, most of the breakpoints are located in regions highly involved in chromosome interactions. Further studies should be carried out to confirm the potential role of chromosome territories’ proximity in promoting translocations, to help understand mechanisms underlying genome structural variation possibly associated to genetic diseases, but also fundamental to promote genome evolution.

Class III peroxidase genes in the moss Dicranum scoparium: Identification and abiotic stress induced expression analysis

Class III peroxidases (POD: EC 1.11.1.7) are classical secretory plant peroxidases belonging to a large multigene family with diverse functions. Members of the POD family have been well-studied and characterized in many plants, including three species of bryophytes, but not from the moss Dicranum scoparium Hedw. Ecologically, D. scoparium is a very important species, which has a widespread distribution throughout the Holarctic. Here we present the first comprehensive report on the POD gene family in D. scoparium, identifying 22 genes encoding PODs (DsPODs), two of which were cloned for verification. All genes were deposited to GenBank under the third-party annotation (Accession numbers TPA: BK061169 – BK061190). Here, we present an in silico study of the physicochemical properties of these proteins. Analyses of conserved domains and subcellular localization suggested that DsPODs have classical peroxidase domain structure; they are secretory proteins and most of them are extracellular. Eight DsPODs highly homologous to Class III peroxidases from the mosses Pohlia nutans and Physcomitrium patens were further microcharacterized. All eight DsPODs possess a haem ligand and active sites necessary for enzymatic activity; they also contained sites for posttranslational modifications. Prediction of secondary structure indicated that these proteins mainly consist of α-helices and random coils. Experiments involving the reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) showed that DsPOD1, DsPOD2, DsPOD6, and DsPOD8 are differentially upregulated in response to stress. The stresses applied here included CdCl2, paraquat, unfavorable temperatures, and a hydration-desiccation-rehydration cycle. Our results indicate that Class III PODs contribute to the abiotic stress tolerance of D. scoparium, and specific DsPOD genes may play diverse roles in the response of the moss to stress.

A mosquito-specific antennal protein is critical for the attraction to human odor in the malaria vector Anopheles gambiae

Mosquitoes rely mainly on the sense of smell to decipher their environment and locate suitable food sources, hosts for blood feeding and oviposition sites. The molecular bases of olfaction involve multigenic families of olfactory proteins that have evolved to interact with a narrow set of odorants that are critical for survival. Understanding the complex interplay between diversified repertoires of olfactory proteins and ecologically-relevant odorant signals, which elicit important behaviors, is fundamental for the design of novel control strategies targeting the sense of smell of disease vector mosquitoes. Previously, large multigene families of odorant receptor and ionotropic receptor proteins, as well as a subset of odorant-binding proteins have been shown to mediate the selectivity and sensitivity of the mosquito olfactory system. In this study, we identify a mosquito-specific antennal protein (MSAP) gene as a novel molecular actor of odorant reception. MSAP is highly conserved across mosquito species and is transcribed at an extremely high level in female antennae. In order to understand its role in the mosquito olfactory system, we generated knockout mutant lines in Anopheles gambiae, and performed comparative analysis of behavioral and physiological responses to human-associated odorants. We found that MSAP promotes female mosquito attraction to human odor and enhances the sensitivity of the antennae to a variety of odorants. These findings suggest that MSAP is an important component of the mosquito olfactory system, which until now has gone completely unnoticed.

The neighbouring genes AvrLm10A and AvrLm10B are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes.

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as "cooperating proteins", able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue.

MAPping tubulin mutations.

Microtubules are filamentous structures that play a critical role in a diverse array of cellular functions including, mitosis, nuclear translocation, trafficking of organelles and cell shape. They are composed of α/β-tubulin heterodimers which are encoded by a large multigene family that has been implicated in an umbrella of disease states collectively known as the tubulinopathies. De novo mutations in different tubulin genes are known to cause lissencephaly, microcephaly, polymicrogyria, motor neuron disease, and female infertility. The diverse clinical features associated with these maladies have been attributed to the expression pattern of individual tubulin genes, as well as their distinct Functional repertoire. Recent studies, however, have highlighted the impact of tubulin mutations on microtubule-associated proteins (MAPs). MAPs can be classified according to their effect on microtubules and include polymer stabilizers (e.g., tau, MAP2, doublecortin), destabilizers (e.g., spastin, katanin), plus-end binding proteins (e.g., EB1-3, XMAP215, CLASPs) and motor proteins (e.g., dyneins, kinesins). In this review we analyse mutation-specific disease mechanisms that influence MAP binding and their phenotypic consequences, and discuss methods by which we can exploit genetic variation to identify novel MAPs.

Research advances on the structure, function and regulation of NIP aquaporins in plants

<p indent="0mm">Aquaporin protein (AQP) family, as a “carrier” of water, micronutrients, and solute macromolecules in plants, plays an important role in material transport. Unlike other members of the AQP family, nodulin 26-like intrinsic proteins (NIPs) mainly penetrate small molecules, such as water, glycerin, silicic acid, boric acid, and urea. In addition, it is involved in plant cell osmoregulation, seed germination, lateral root production, leaf and flower growth and development, and response to biotic and abiotic (drought, chilling, salinity, metalloid toxicity, hypoxia, etc.) stresses. So far, structures of the NIP family have been partially reported. The NIPs can be divided into three classes based on the pore size of the ar/R selective filter, which determines the absorption and transportation of different substrates. Moreover, the NIPs contain two NPA motifs, which are located in loop B and loop E and have a typical ring-like hourglass structure. Therefore, different structures of the NIP subfamily play different roles in plant membrane solute transport and water balance. The <italic>NIP</italic> gene family has been identified in <italic>Arabidopsis thaliana</italic>,<italic> Oryza sativa</italic>, and other plants, whose function needs to be achieved through a variety of signaling pathways and the synergistic effect of physiological processes. In recent years, more and more reports have shown how the <italic>NIP</italic> gene participates in regulating gene expression levels under various stress conditions and interacts with a variety of stress proteins to jointly regulate the water and osmotic balance of plants and improve plant resistance and adaptability. The co-expression of <italic>NIPs</italic> with <italic>AtBOR1</italic>, <italic>AtNodGS</italic>, and <italic>AtACR3</italic> genes can enhance the transport of solute and aquaporin. Besides, the interaction between NIP and 14-3-3f proteins can stabilize cell structure and substance synthesis in vivo, thus enhancing plant cold resistance. Interestingly, <italic>NIP</italic> is regulated by WRKY transcription factors and plays an active role in the regulation and distribution of metalloid elements in plants. In addition, the <italic>NIP </italic>genes are involved in the response of various hormone signaling pathways in plants, and their gating mechanisms and osmotic stress responses are regulated by the C- and N-terminal phosphorylation sites of NIP proteins, but the specific response mechanisms remain to be further explored. <italic>NIPs</italic> are a large multi-gene family with functional redundancy among members, and the similarities and differences in functions and mechanisms of different subfamilies of <italic>NIP</italic> genes need to be investigated. One of our main subsequent research directions is to explore the stress resistance network of the <italic>NIP</italic> genes, especially their response mechanisms to biotic stress. What’s more, NIPs can interact with PIP proteins belonging to the AQP family and affect the diffusion of water molecules by changing their positions. Therefore, whether NIPs interact with other members of the AQP family, such as SIPs and TIPs, deserves to be validated further. In summary, the origin and evolution, structure, classification, biological function, and regulation mechanism of the NIP family in plants are reviewed, which provides new insights and references for further research.

Trans-cyclosulfamidate mannose-configured cyclitol allows isoform-dependent inhibition of GH47 α-d-mannosidases through a bump-hole strategy.

Class I inverting exo-acting α-1,2-mannosidases (CAZY family GH47) display an unusual catalytic itinerary featuring ring-flipped mannosides, 3S1 → 3H4‡ → 1C4. Conformationally locked 1C4 compounds, such as kifunensine, display nanomolar inhibition but large multigene GH47 mannosidase families render specific "isoform-dependent" inhibition impossible. Here we develop a bump-and-hole strategy in which a new mannose-configured 1,6-trans-cyclic sulfamidate inhibits α-d-mannosidases by virtue of its 1C4 conformation. This compound does not inhibit the wild-type GH47 model enzyme by virtue of a steric clash, a "bump", in the active site. An L310S (a conserved residue amongst human GH47 enzymes) mutant of the model Caulobacter GH47 awoke 574 nM inhibition of the previously dormant inhibitor, confirmed by structural analysis of a 0.97 Å structure. Considering that L310 is a conserved residue amongst human GH47 enzymes, this work provides a unique framework for future biotechnological studies on N-glycan maturation and ER associated degradation by isoform-specific GH47 α-d-mannosidase inhibition through a bump-and-hole approach.

Tubby-like proteins (TLPs) transcription factor in different regulatory mechanism in plants: a review.

Tubby-like proteins (TLPs) transcription factors are found in single-celled to multi-cellular eukaryotes in the form of large multigene families. TLPs are identified through a specific signature of carboxyl terminal tubby domain, required for plasma membrane tethering and amino terminal F-box domain communicate as functional SCF-type E3 ligases. The comprehensive distribution of TLP gene family members in diverse species indicates some conserved functions of TLPs in multicellular organisms. Plant TLPs have higher gene members than animals and these members reported important role in multiple physiological and developmental processes and various environmental stress responses. Although the TLPs are suggested to be a putative transcription factors but their functional mechanism is not much clear. This review provides significant recent updates on TLP-mediated regulation with an insight into its functional roles, origin and evolution and also phytohormones related regulation to combat with various stresses and its involvement in adaptive stress response in crop plants.

Functional Divergence in a Multi-gene Family Is a Key Evolutionary Innovation for Anaerobic Growth in Saccharomyces cerevisiae.

The amplification and diversification of genes into large multi-gene families often mark key evolutionary innovations, but this process often creates genetic redundancy that hinders functional investigations. When the model budding yeast Saccharomyces cerevisiae transitions to anaerobic growth conditions, the cell massively induces the expression of seven serine/threonine-rich anaerobically-induced cell wall mannoproteins (anCWMPs): TIP1, TIR1, TIR2, TIR3, TIR4, DAN1, and DAN4. Here, we show that these genes likely derive evolutionarily from a single ancestral anCWMP locus, which was duplicated and translocated to new genomic contexts several times both prior to and following the budding yeast whole genome duplication (WGD) event. Based on synteny and their phylogeny, we separate the anCWMPs into four gene subfamilies. To resolve prior inconclusive genetic investigations of these genes, we constructed a set of combinatorial deletion mutants to determine their contributions toward anaerobic growth in S. cerevisiae. We found that two genes, TIR1 and TIR3, were together necessary and sufficient for the anCWMP contribution to anaerobic growth. Overexpressing either gene alone was insufficient for anaerobic growth, implying that they encode non-overlapping functional roles in the cell during anaerobic growth. We infer from the phylogeny of the anCWMP genes that these two important genes derive from an ancient duplication that predates the WGD event, whereas the TIR1 subfamily experienced gene family amplification after the WGD event. Taken together, the genetic and molecular evidence suggests that one key anCWMP gene duplication event, several auxiliary gene duplication events, and functional divergence underpin the evolution of anaerobic growth in budding yeasts.

Non-canonical odor coding in the mosquito

SUMMARYAedes aegypti mosquitoes are a persistent human foe, transmitting arboviruses including dengue when they feed on human blood. Mosquitoes are intensely attracted to body odor and carbon dioxide, which they detect using ionotropic chemosensory receptors encoded by three large multi-gene families. Genetic mutations that disrupt the olfactory system have modest effects on human attraction, suggesting redundancy in odor coding. The canonical view is that olfactory sensory neurons each express a single chemosensory receptor that defines its ligand selectivity. We discovered that Ae. aegypti uses a different organizational principle, with many neurons co-expressing multiple chemosensory receptor genes. In vivo electrophysiology demonstrates that the broad ligand-sensitivity of mosquito olfactory neurons depends on this non-canonical co-expression. The redundancy afforded by an olfactory system in which neurons co-express multiple chemosensory receptors may increase the robustness of the mosquito olfactory system and explain our long-standing inability to disrupt the detection of humans by mosquitoes.

Integrated viral elements suggest the dual lifestyle of Tetraselmis spp. Polinton-like viruses.

In this study, we aimed at exploring horizontal gene transfer between viruses and Chlorodendraceae green algae (Chlorophyta) using available genomic and transcriptomic sequences for twenty algal strains. We identified a significant number of genes sharing a higher sequence similarity with viral homologues, thus signalling their possible involvement in horizontal gene transfers with viruses. Further characterization showed that many of these genes were clustered in DNA regions of several tens to hundreds of kilobases in size, originally belonging to viruses related to known Tetraselmis spp. viruses (TetV and TsV). In contrast, the remaining candidate HGT genes were randomly dispersed in the algal genomes, were more frequently transcribed, and belonged to large multigene families. The presence of homologues in Viridiplantae suggested that the latter were more likely of algal rather than viral origin. We found a remarkable diversity in polinton-like virus (PLV) elements inserted in Tetraselmis genomes, all of which were most similar to the Tetraselmis striata virus (TsV). The genes of PLV elements are transcriptionally inactive with the notable exception of the homologue of the TVSG_00024 gene of TsV whose function is unknown. We suggest that this gene may be involved in a sentinel process to trigger virus reactivation and excision in response to an environmental stimulus. Altogether, these results provide evidence that TsV-related viruses have a dual lifestyle, alternating between a free viral phase (i.e. virion) and a phase integrated into host genomes.

Vivax Malaria and the Potential Role of the Subtelomeric Multigene vir Superfamily.

Vivax malaria, caused by Plasmodium vivax, remains a public health concern in Central and Southeast Asia and South America, with more than two billion people at risk of infection. Compared to Plasmodium falciparum, P. vivax is considered a benign infection. However, in recent decades, incidences of severe vivax malaria have been confirmed. The P. falciparum erythrocyte membrane protein 1 family encoded by var genes is known as a mediator of severe falciparum malaria by cytoadherence property. Correspondingly, the vir multigene superfamily has been identified as the largest multigene family in P. vivax and is implicated in cytoadherence to endothelial cells and immune response activation. In this review, the functions of vir genes are reviewed in the context of their potential roles in severe vivax malaria.

Functional Diversity of the Lepidopteran ATP-Binding Cassette Transporters.

The ATP-binding cassette (ABC) transporter gene family is ubiquitous in the living world. ABC proteins bind and hydrolyze ATP to transport a myriad of molecules across various lipid-containing membrane systems. They have been studied well in plants for transport of a variety of compounds and particularly, in vertebrates due to their direct involvement in resistance mechanisms against several toxic molecules/metabolites. ABC transporters in insects are found within large multigene families involved in the efflux of chemical insecticides and toxic/undesired metabolites originating from food and endogenous metabolism. This review deals with ABC transporter subfamilies of few agronomically important Lepidopteran pests. The transcriptional dynamics and regulation of ABC transporters during insect development emphasizes their functional diversity against insecticides, Cry toxins, and plant specialized metabolites. To generate insights about molecular function and physiological roles of ABCs, functional and structural characterization is necessary. Also, expansion and divergence of ABC transporter gene subfamilies in Lepidopteran insects needs more systematic investigation. We anticipate that newer methods of insect control in agriculture can benefit from an understanding of ABC transporter interactions with a vast range of natural specialized molecules and synthetic compounds.

Chromodomain Protein Interacts with H3K9me3 and Controls RBC Rosette Formation by Regulating the Expression of a Subset of RIFINs in the Malaria Parasite

Plasmodium falciparum expresses clonally variant proteins on the surface of infected erythrocytes to evade the host immune system. The clonally variant multigene families include var, rifin, and stevor, which express Erythrocyte Membrane Protein 1 (EMP1), Repetitive Interspersed Families of polypeptides (RIFINs), and Sub-telomeric Variable Open Reading frame (STEVOR) proteins, respectively. The rifins are the largest multigene family and are essentially involved in the RBC rosetting, the hallmark of severe malaria. The molecular regulators that control the RIFINs expression in Plasmodium spp. have not been reported so far. This study reports a chromodomain-containing protein (PfCDP) that binds to H3K9me3 modification on P. falciparum chromatin. Conditional deletion of the chromodomain (CD) gene in P. falciparum using an inducible DiCre-LoxP system leads to selective up-regulation of a subset of virulence genes, including rifins, a few var, and stevor genes. Further, we show that PfCDP conditional knockout (PfΔCDP) promotes RBC rosette formation. This study provides the first evidence of an epigenetic regulator mediated control on a subset of RIFINs expression and RBC rosetting by P. falciparum.

Sheep Infection Trials with 'Phase-Locked' Vpma Expression Variants of Mycoplasma agalactiae-Towards Elucidating the Role of a Multigene Family Encoding Variable Surface Lipoproteins in Infection and Disease.

The significance of large multigene families causing high-frequency surface variations in mycoplasmas is not well-understood. Previously, VpmaY and VpmaU clonal variants of the Vpma family of lipoproteins of M. agalactiae were compared via experimental sheep infections using the two corresponding ‘Phase-Locked Mutants’. However, nothing is known about the infectivity of the remaining four Vpma expression variants VpmaX, VpmaW, VpmaZ and VpmaV as they were never evaluated in vivo. Here, in vivo infection and disease progression of all six Vpma expressers constituting the Vpma family of type strain PG2 were compared using the corresponding xer1-disrupted PLMs expressing single well-characterized Vpmas. Each of the six PLMs were separately evaluated using the intramammary sheep infection model along with the control phase-variable wildtype strain PG2. Thorough bacteriological, pathological and clinical examinations were performed, including assessment of milk quality, quantity and somatic cell counts. Altogether, the results indicated that the inability to vary the Vpma expression phase does not hamper the initiation of infection leading to mastitis for all six PLMs, except for PLMU, which showed a defect in host colonization and multiplication for the first 24 h p.i. and pathological/bacteriological analysis indicated a higher potential for systemic spread for PLMV and PLMX. This is the first study in which all isogenic expression variants of a large mycoplasma multigene family are tested in the natural host.

A single point mutation in a member of FAD2 multigene family resulted in the generation of a high oleic line of Silybum marianum (L.) Gaertn.

Silybum marianum (L.) Gaertn. has been recently valorised as oilseed crop for its productivity and for the chemical composition of its oil. However, a high oleic acid oil (HO) line, which would be useful for cooking but also for biodiesel production and other industrial applications, was not found in its natural germplasm. To address this issue, an ethyl methanesulfonate-induced mutagenesis programme was implemented, and the screening of the mutagenized population led to the identification of a HO mutant line with a much higher oleic acid oil content with respect to the wild type (WT). Since Fatty Acid Desaturase 2 (FAD2) genes are responsible for the determination of oleic acid levels in vegetable oils of other species, the present research focused on the characterization of this gene family as the possible target of mutagenesis. An extremely large multigene family consisting of twenty-one unique and complete FAD2 orthologous sequences was identified in S. marianum. Sequence comparison of all the SmFAD2 genes between the WT and HO genotypes revealed a single nucleotide polymorphism (SNP) in SmFAD2–1, the only fruit-specific gene, resulting in a non-synonymous substitution in the predicted protein in the high oleic genotype. The SNP and the HO phenotype were found to perfectly cosegregate in the F2 progeny derived from the HO x WT crosses, suggesting the direct implication of this gene in the determination of oleic and linoleic acid fruit content in S. marianum fruits.

A peroxidase purified from cowpea roots possesses high thermal stability and displays antifungal activity against Colletotrichum gloeosporioides and Fusarium oxysporum

Plants encode specific class III peroxidases (POX) as large multigene families that are involved in lignification, cell elongation, stress defense, and seed germination. However, many functions associated with these plant enzymes are not fully understood. Here, a class III peroxidase (Vu-RPOX) was purified from cowpea (Vigna unguiculata) roots with 0–90% ammonium sulfate precipitation, chitin column and Resource Q anion-exchange chromatography. Vu-RPOX is a glycoprotein composed by a 58 kDa polypeptide chain and pI of 3.79. Two peptide sequences of Vu-RPOX (RGLDVVNDIKT and RCSTFINRL) generated by Nano ESI-Q-TOF MS/MS analysis showed variable identity with seed coat peroxidase of Glycine max, peroxidase E5 of Vigna radiata, hypothetical peroxidase of Phaseolus vulgaris, and peroxidase E5-like of Vigna unguiculata. Vu-RPOX follows the Michaelis-Menten kinetics, optimal activity at pH 6.0, and was high thermo-stable when incubated up to 75 °C for 90 min. The catalytic activity of Vu-RPOX decreased in the presence of the classic peroxidase inhibitors, but was not inhibited by MgCl2, NH4Cl, MnCl2, CaCl2, and NaCl. Vu-RPOX delay the vegetative growth of the phytopathogenic fungi Colletrotrichum gloeosporioides and Fusarium oxysporum. Based on its properties Vu-RPOX is a potentially useful molecule for application in biomedical, biotechnological, industrial, and agricultural areas particularly because it showed high stability when exposed to a wide range of temperatures and performance at pH 4–7 and impairment of mycelial growth of C. gloeosporioides and F. oxysporum.