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• RNAi in the phytoplasma vector Euscelidius variegatus is long-lasting. • Silencing of ATP synthase β transcript reduces expression of the cognate protein. • dsRNA injection generates siRNAs mapping only on the fragment used to design dsRNAs. • Silenced insects acquire chrysanthemum yellows and Flavescence dorée phytoplasmas. • Silencing of ATP synthase β reduces the multiplication of both phytoplasmas. The leafhopper Euscelidius variegatus is a natural vector of the chrysanthemum yellows phytoplasma (CYp) and a laboratory vector of the Flavescence dorée phytoplasma (FDp). Previous studies indicated a crucial role for insect ATP synthase α and β subunits during phytoplasma infection of the vector species. Gene silencing of ATP synthase β was obtained by injection of specific dsRNAs in E. variegatus. Here we present the long-lasting nature of such silencing, its effects on the small RNA profile, the significant reduction of the corresponding protein expression, and the impact on phytoplasma acquisition capability. The specific transcript expression was silenced at least up to 37 days post injection with an average reduction of 100 times in insects injected with dsRNAs targeting ATP synthase β (dsATP) compared with those injected with dsRNAs targeting green fluorescent protein (dsGFP), used as negative controls. Specific silencing of this gene was also confirmed at protein level at 15 days after the injection. Total sRNA reads mapping to dsATP and dsGFP sequences in analysed libraries showed in both cases a peak of 21 nt, a length consistent with the generation of dsRNA-derived siRNAs by RNAi pathway. Reads mapped exclusively to the fragment corresponding to the injected dsATPs, probably indicating the absence of a secondary machinery for siRNA synthesis. Insects injected either with dsATP or dsGFP successfully acquired CYp and FDp during feeding on infected plants. However, the average phytoplasma amount in dsATP insects was significantly lower than that measured in dsGFP specimens, indicating a probable reduction of the pathogen multiplication when ATP synthase β was silenced. The role of the insect ATP synthase β during phytoplasma infection process is discussed. [ABSTRACT FROM AUTHOR]

Molecular mechanisms underlying phytoplasma interactions with host plants are largely unknown. In this study attempts were made to identify effectors of three phytoplasma strains related to ‘ Ca. P. aurantifolia’, crotalaria phyllody (CrP), faba bean phyllody (FBP), and witches’ broom disease of lime (WBDL), using information from draft genome of peanut witches’ broom phytoplasma. Seven putative effectors were identified in WBDL genome (SAP11, SAP21, Eff64, Eff115, Eff197, Eff211 and EffSAP67), five (SAP11, SAP21, Eff64, Eff99 and Eff197) in CrP and two (SAP11, Eff64) in FBP. No homologs to Eff64, Eff197 and Eff211 in phytoplasmas of other phylogenetic groups were found. SAP11 and Eff64 homologs of ‘ Ca. P. aurantifolia’ strains shared at least 95.9% identity and were detected in the three phytoplasmas, supporting their role within the group. Five of the putative effectors (SAP11, SAP21, Eff64, Eff115, and Eff99) were transcribed from total RNA extracts of periwinkle plants infected with these phytoplasmas. Transcription profiles of selected putative effectors of CrP, FBP and WBDL indicated that SAP11 transcripts were the most abundant in the three phytoplasmas. SAP21 transcript levels were comparable to those of SAP11 for CrP and not measurable for the other phytoplasmas. Eff64 had the lowest transcription level irrespective of sampling date and phytoplasma isolate. Eff115 transcript levels were the highest in WBDL infected plants. This work reports the first sequence information for 14 putative effectors in three strains related to ‘ Ca. P. aurantifolia’, and offers novel insight into the transcription profile of five of them during infection of periwinkle. [ABSTRACT FROM AUTHOR]

Phytoplasmas are phloem-limited plant pathogenic bacteria causing diseases in many plant species. They are transmitted by Hemipteran insect species in a persistent-propagative manner. Phytoplasmas are wall-less, and their membrane proteins are involved in pathogen internalization into host cells. We focused on the immunodominant membrane protein (Imp) of Flavescence dorée phytoplasma (FDp), a grapevine quarantine pest and a major threat to European viticulture. Scaphoideus titanus is the main natural vector of FDp to grapevine, whereas Euscelidius variegatus is commonly used as laboratory vector. Previous works indicated that recombinant Imp of two FDp strains (FD-C and FD-D) selectively interact with gut proteins from vector species rather than those from non-vectors. Here, similar patterns of interacting insect gut proteins were obtained from both vector species, following pull-down with His-tagged FDp Imps. After identification of several targets, four S. titanus and five E. variegatus proteins interacting with Imp were further characterized by measuring expression in different insect tissues and in healthy vs. infected insects. Specific RNAi silencing of two of these vector genes, namely natterin and legumain, resulted in a significant reduction of phytoplasma multiplication in insects upon pathogen acquisition, compared to control insects. Natterin displays a DM9 domain and legumain possesses a signature of G protein receptor, supporting their involvement as FDp Imp receptors. Outcomes of this work are discussed with particular attention devoted to the gain of knowledge on host/pathogen interaction as well as to the potential impact on improvement phytoplasma disease management. • Grapevine Flavescence dorée phytoplasma (FDp) is a severe quarantine plant disease. • Recombinant Imp proteins of FDp-C and D strains interact with insect gut elements. • RNAi silencing of insect natterin and legumain reduces phytoplasma load in vectors. • They have features of protein interaction mediating pathogen colonization of vector. • Natterin and legumain may work as pathogen receptors in gut of insect vectors. [ABSTRACT FROM AUTHOR]

Fungi living in heavy metal polluted soils have evolved different cellular and molecular systems to adapt and survive in these harsh environments. Oidiodendron maius Zn is an ericoid mycorrhizal fungus previously shown to be highly tolerant to zinc thanks to antioxidative enzymes and membrane transporters. A novel gene, OmFCR1, was recently identified from this fungus because it conferred strong cadmium tolerance when expressed in yeast. OmFCR1 codes for a protein belonging to the PLAC8 family and physically interacts in yeast with the mismatch repair system, involved in DNA damage repair. The O. maius Zn genome also contains another gene - named OmFCR2 - that codes for a protein sharing with OmFCR1, the PLAC8 domain and other sequence similarities. In this work, we analyzed gene expression of OmFCR1 and OmFCR2 in the fungus O. maius Zn when exposed to cadmium, the ability of OmFCR2 to confer cadmium tolerance when expressed in yeast, and the growth of OmFCR1-null mutants of O. maius Zn upon cadmium exposure. Although OmFCR2 was also able to confer some cadmium tolerance to yeast, the different expression pattern of these two genes would suggest different roles in O. maius Zn. [ABSTRACT FROM AUTHOR]

Abstract The biological bases of recovery of two grapevine cultivars, Nebbiolo and Barbera, showing different susceptibility and recovery ability to "flavescence dorée" (FD) phytoplasma infection were investigated. The expression over one vegetative season, in FD-recovered and healthy grapevines, of 18 genes involved in defence, hydrogen peroxide and hormone production was verified at two time points. Difference (Δ) between the relative expressions of August and July were calculated for each target gene of both cultivars. The significance of differences among groups assessed by univariate and multivariate statistical methods, and sPLS-DA analyses of the Δ gene expression values, showed that control and recovered grapevines of both cultivars were clearly separated. The Barbera-specific deregulation of defence genes supports a stronger response of this variety, within a general frame of interactions among H 2 O 2 , jasmonate and ethylene metabolisms, common to both varieties. This may strengthen the hypothesis that FD-recovered Barbera grapevines modulate transcription of their genes to cope with potential damages associated to the alteration of their oxidative status. Nebbiolo variety would fit into this picture, although with a less intense response, in line with its lower degree of susceptibility and recovery incidence to FD, compared to Barbera. The results evidenced a scenario where plant response to phytoplasma infection is highly affected by climatic and edaphic conditions. Nevertheless, even after several years from the original FD infection, it was still possible to distinguish, at molecular level, control and recovered grapevines of both cultivars by analyzing their overall-season response, rather than that of a single time point. [ABSTRACT FROM AUTHOR]