Frequency of a natural truncated allele of <Emphasis Type="Italic">MdMLO19</Emphasis> in the germplasm of <Emphasis Type="Italic">Malus domestica</Emphasis>

Podosphaera leucotricha is the causal agent of powdery mildew (PM) in apple. To reduce the amount of fungicides required to control this pathogen, the development of resistant apple cultivars should become a priority. Resistance to PM was achieved in various crops by knocking out specific members of the MLO gene family that are responsible for PM susceptibility (S-genes). In apple, the knockdown of MdMLO19 resulted in PM resistance. However, since gene silencing technologies such as RNAi are perceived unfavorably in Europe, a different approach that exploits this type of resistance is needed. This work evaluates the presence of non-functional naturally occurring alleles of MdMLO19 in apple germplasm. The screening of the re-sequencing data of 63 apple individuals led to the identification of 627 single nucleotide polymorphisms (SNPs) in five MLO genes (MdMLO5, MdMLO7, MdMLO11, MdMLO18, and MdMLO19), 127 of which were located in exons. The T-1201 insertion of a single nucleotide in MdMLO19 caused the formation of an early stop codon, resulting in a truncated protein lacking 185 amino acids, including the calmodulin-binding domain. The presence of the insertion was evaluated in 115 individuals. It was heterozygous in 64 and homozygous in 25. Twelve of the 25 individuals carrying the insertion in homozygosity were susceptible to PM. After barley, pea, cucumber, and tomato, apple would be the fifth species for which a natural non-functional mlo allele has been found.     

Protein–nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein–DNA and protein–ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein–DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced ³¹P–¹³C polarization-transfer experiments followed by the recording of a 2D ¹³C–¹³C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.     

Animal xenodiversity in Italian inland waters: distribution, modes of arrival, and pathways

The paper provides a list of the non-indigenous animal species occurring today in Italian inland waters. Xenodiversity was found to amount to 112 species (64 invertebrates and 48 vertebrates), which contribute for about 2% to the inland-water fauna in Italy. Northern and central regions are most affected, and Asia, North America, and the rest of Europe are the main donor continents. The large majority of non-indigenous species entered Italy as a direct or indirect effect of human intervention. A difference between invertebrates and vertebrates was found for their mode of arrival (unintentional for invertebrates and intentional for vertebrates). Accidental transport, in association with both fish (for aquaculture or stock enhancement) and crops, has been the main vector of invertebrate introductions, whereas vertebrates were mostly released for stocking purposes. Overall stock enhancement (47.92%) and culture (37.5%) prevailed over the other pathways. Seventeen and 7 species of our list are included among the 100 worst invasive species of Europe (DAISIE) and of the world (IUCN), respectively. For some (but not all) non-indigenous species recorded in Italy the multilevel impact exerted on the recipient communities and ecosystems is known, even if rarely quantified, but knowledge on their chronic impact is still missing. Additional research is needed to provide criteria for prioritizing intervention against well established invaders and identify which new potential invader should be targeted as “unwanted”.     

Stringent requirement for HRD1, SEL1L,and OS-9/XTP3-B for disposal of ERAD-LS substrates

Sophisticated quality control mechanisms prolong retention of protein-folding intermediates in the endoplasmic reticulum (ER) until maturation while sorting out terminally misfolded polypeptides for ER-associated degradation (ERAD). The presence of structural lesions in the luminal, transmembrane, or cytosolic domains determines the classification of misfolded polypeptides as ERAD-L, -M, or -C substrates and results in selection of distinct degradation pathways. In this study, we show that disposal of soluble (nontransmembrane) polypeptides with luminal lesions (ERAD-L_S substrates) is strictly dependent on the E3 ubiquitin ligase HRD1, the associated cargo receptor SEL1L, and two interchangeable ERAD lectins, OS-9 and XTP3-B. These ERAD factors become dispensable for degradation of the same polypeptides when membrane tethered (ERAD-L_M substrates). Our data reveal that, in contrast to budding yeast, tethering of mammalian ERAD-L substrates to the membrane changes selection of the degradation pathway.     

Oxidative damage and antioxidant defense system in leaves of Vicia faba major L. cv. Bartom during soil flooding and subsequent drainage

The adaptive reactions of Vicia faba major L. cv. Bartom to 13-27 days soil flooding and to 14 days of drainage following 13-days of soil flooding were studied. Under flooding, oxygen diffusion rate (ODR) in the root zone decreased from 2.28–3.44 to 0.09–0.28?µmol O₂ m^?2 s^?1; the soil redox potential (Eh) decreased from 543 to 70 mV. Upon drainage of flooded soil the ODR and Eh values returned to the control levels. Oxidative damage and defense systems in leaves were assessed by the concentration of thiobarbituric acid reactive substances (TBARs) and by the activities of superoxide dismutase (SOD) and glutathione reductase (GR). Two stages of stress development are described. During the first stage (1–13 days) shoot dry mass did not decrease, the TBARs concentration and SOD activity increased, the GR activity decreased. The second stage (13–27 days) was characterized by a decrease in the TBARs concentration, SOD and GR activities, pigment concentrations and shoot dry mass. Drainage of flooded soil resulted in elevated concentrations of TBARs and also increased the activities of SOD and GR. Increased SOD activity in the first stage of hypoxic stress development and activations of SOD and GR at oxygen re-entry to soil are responsible for tolerance of Vicia faba to hypoxic and post hypoxic stress associated with soil flooding and subsequent drainage.     

A PCR-based diagnostic tool for distinguishing grape skin color mutants

Berry skin color mutants are phenotypically different from their original cultivars, but they show identical molecular profile if analysed by using microsatellite markers. This work gives an easy, inexpensive and quick diagnostic tool to discriminate these somatic variants. We distinguished some grape (Vitis vinifera L.) skin color mutants from white to red or pink and from black to grey, pink or white and we investigated their molecular bases by single-strand conformational polymorphism (SSCP), single base primer extension and coding sequence analysis of anthocyanin biosynthetic enzyme genes and by polymerase chain reaction (PCR) analysis of VvmybA1 regulatory gene. Analyses of structural genes did not reveal polymorphisms between wild type and mutant cultivars but only among different varieties, whereas the study of VvmybA1 regulatory gene has given important outcomes for color mutants characterisation. The discrimination between white wild type and its derived colored mutant and between black wild type and white mutant has been obtained through a simple test of amplification for presence/absence. The discrimination between black wild type and less colored mutant has occurred through a quantitative result on agarose gel confirmed by real-time PCR analysis: the amount of functional allele in less colored somatic variants genome was about one-fourth of the correspondent quantity in original black cultivars genome.     

GarA is an essential regulator of metabolism in Mycobacterium tuberculosis

Alpha‐ketoglutarate is a key metabolic intermediate at the crossroads of carbon and nitrogen metabolism, whose fate is tightly regulated. In mycobacteria the protein GarA regulates the tricarboxylic acid cycle and glutamate synthesis by direct binding and regulation of three enzymes that use α‐ketoglutarate. GarA, in turn, is thought to be regulated via phosphorylation by protein kinase G and other kinases. We have investigated the requirement for GarA for metabolic regulation during growth in vitro and in macrophages. GarA was found to be essential to Mycobacterium tuberculosis, but dispensable in non‐pathogenic Mycobacterium smegmatis. Disruption of garA caused a distinctive, nutrient‐dependent phenotype, fitting with its proposed role in regulating glutamate metabolism. The data underline the importance of the TCA cycle and the balance with glutamate synthesis in M. tuberculosis and reveal vulnerability to disruption of these pathways.