Characterization of community structure of culturable endophytic fungi in sweet cherry composite trees and their growth-retarding effect against pathogens.

Endophytic fungi have the potential to protect their host plants in stress situations. Characterizing the ecology and complex interaction between these endophytes and their host plants is therefore of great practical importance, particularly in horticultural plants. Among horticultural plants, fruit trees form a special category because of their longevity and because they are composites of rootstock and scion, which often belong to different plant species. Here we present the first characterization of culturable endophytic fungal community of sweet cherry. Samples from the Hungarian cultivar 'Petrus' grafted on 11 different rootstocks were collected in autumn and in spring in a bearing orchard and the dependence of colonization rate and endophyte diversity on rootstock, organ and season was analysed. On the basis of their ITS sequences 26 fungal operational taxonomic units were identified at least down to the genus level. The dominant genus, comprising more than 50% of all isolates, was Alternaria, followed by different Fusarium and Epicoccum species. We observed some organ-specificity amongst endophytes, and organs showed more sizeable differences in colonization rates and endophyte diversity than rootstocks. Most dynamic endophyte populations, strongly influenced by environmental conditions and crop management, were observed in leaves. The potential of selected endophytes to confer protection against Monilinia laxa was also analysed and 7 isolates were found to inhibit the growth of this pathogen in vitro.

The B″ regulatory subunit of protein phosphatase 2A mediates the dephosphorylation of rice retinoblastoma-related protein-1.

The phosphorylation of plant retinoblastoma-related (RBR) proteins by cyclin-dependent kinases (CDKs) is well documented, but the counteracting phosphatases have not been identified yet. We report here that rice retinoblastoma-related protein-1 (OsRBR1) interacted with the B″ subunit of rice protein phosphatase 2A (OsPP2A B″) and underwent reversible phosphorylation during the cell division cycle. The OsRBR1-OsPP2A B" association required B domain in OsRBR1 and the C-terminal region of OsPP2A B″. We found by immunoprecipitation that OsPP2A B″, OsPP2A catalytic subunit subtype II, PSTAIRE-type CDK and OsRBR1 were in the same protein complex, indicating a physical association between the phosphatase, the kinase and their common substrate. OsPP2A B″ contains three predicted CDK phosphorylation sites: Ser95, Ser102 and Ser119. The in vitro phosphorylation of Ser95 and Ser119 with PSTAIRE-kinases was verified by mass spectrometry. We generated a series of phosphorylation site mutants to mimic the dephosphorylated or phosphorylated states of OsPP2A B″, and confirmed that all of the three predicted sites can be phosphorylated. Yeast two-hybrid experiments suggested that the phosphorylation of OsPP2A B″ promoted the formation of the OsPP2A holoenzyme. A triple phosphorylation mimicking OsPP2A B″ mutant containing holoenzyme showed higher activity in phosphatase assays. Our data collectively show that the phosphatase activity of OsPP2A against OsRBR1 is regulated by the phosphorylation of its B″ regulatory subunit. However, the analysis of the effect of okadaic acid, a phosphatase inhibitor, in rice cell suspension cultures revealed that the dephosphorylation of OsRBR1 was completely inhibited only by high dose (300 nM) of the okadaic acid during the cell cycle progression. Therefore the role of the protein phosphatase 1 should be considered as an additional post translational regulatory component of RBR protein function in higher plants.

The genome of Beet cryptic virus 1 shows high homology to certain cryptoviruses present in phylogenetically distant hosts.

UNLABELLED: This study determined the complete nucleotide sequence of Beet cryptic virus 1 (BCV1). As expected by analogy to previously sequenced alphacryptoviruses, dsRNA1 (2008 bp) encodes a 72.5-kDa protein containing sequence motifs characteristic for RNA-dependent RNA polymerases (RdRp). In addition to the full-length dsRNA1, a truncated form was also detected in dsRNA extracts. dsRNA2 (1783 bp) codes for the viral coat protein (CP) as proven by the identity of the predicted CP sequence to peptide sequences of the purified virion protein. The amino acid sequence of BCV1 RdRp as well as the 5'- and 3'-UTRs show 81-85% identity to the corresponding regions of Vicia cryptic virus (VCV), White clover cryptic virus 1 (WCCV1) and Carrot cryptic virus (CaCV). The amino acid sequence identity of the CP is about 55-62%, moreover, a strong conservation of predicted alpha-helical regions was observed. The high degree of similarity of these seed- and pollen-transmitted viruses persisting in phylogenetically distant hosts, together with their high similarity to fungal partitiviruses strongly supports the hypothesis that horizontal transfer by a fungus played a role in the emergence of the present cryptovirus species. The change in the distribution of cryptic viruses may also be due to human influence: While earlier BCV1 occurred frequently in sugar beet cultivars, it is very rare in cultivars currently used in agricultural practice and was detected in only one of the 28 cultivars investigated in our experiments. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11262-009-0432-4) contains supplementary material, which is available to authorized users.

Targeting dsRNA-specific single-chain Fv antibody fragments to different cellular locations in Nicotiana tabacum L.

Expression of antibodies or antibody fragments in plants is a useful tool for producing active antibody derivatives for diagnostic or pharmaceutical purposes as well as for immunomodulation. We investigated the effect of cellular expression site on the stability and yield of double-stranded RNA (dsRNA)-specific single-chain Fv-fragments (scFv) in transgenic tobacco. Two antibodies (J2 and P6) belonging to the V23(J558) heavy chain variable gene family but differing in the light chain variable domain were used. scFvs were targeted to the cytoplasm - with or without anchoring them in the plasma membrane -, into the endoplasmic reticulum (ER) and to the apoplast. Although high mRNA concentrations were detected in all cases, scFv proteins accumulated only when scFvs were made ER-resident by appropriate signal sequences. When the ER retention signal was removed to allow scFv-secretion to the apoplast, no scFv-proteins were detected. Despite the strong homology of the VH-sequences of J2 and P6 antibodies, only P6 provided a stable scFv scaffold for intracytoplasmic expression. J2-scFv could not be stabilised either by adding a C-terminal stabilisation signal or by anchoring the protein on the cytoplasmic side of the plasma membrane (PM). It was found that dsRNA-specific J2-scFvs are active in vivo and enhance Potato Virus Y induced symptoms in infected tobacco. This is the first report describing the expression and biological effect of RNA-specific antibodies in plants.

Searching for a new putative cryptic virus in Pinus sylvestris L.

Double-stranded RNAs (dsRNAs) were detected in different pine populations in Germany and Hungary. Two dsRNA species of 1.5 and 1.58 kbp, respectively, persisted in the same trees for at least 2 years and their presence was not associated with any symptoms. The dsRNAs were found to sediment in the VLP (virus-like particles) fraction and to be protected by protein(s) against RNase A digestion at low salt. cDNA cloning and sequencing of the smaller segment (dsRNA2) led to the identification of a putative RNA-dependent RNA-polymerase (RdRp) containing the GDD, as well as three other, conserved motifs. Sequence comparison with different RNA viruses and phylogenetic analysis indicates that the putative RdRp from pine shows highest similarity to the homologous proteins of Beet cryptic virus 3 and of a cryptic virus of Pyrus pyrifolia. On the basis of these results we suggest that the 1.5 and 1.58 kbp dsRNAs in P. sylvestris may represent the genomic segments of a new plant cryptic virus, Cryptoviruses have not yet been reported to occur in Gymnosperms.