First Report of Cymbidium mosaic virus and Odontoglossum ringspot virus in Orchids in Mexico.

In 2010, a survey for viral diseases in commercial, orchid-producing greenhouses was carried out in Morelos, Mexico. Many symptomatic plants were observed. The most common leaf symptoms were yellow mottle, yellow streaks, and chlorotic and necrotic ringspots. Leaf samples were collected from eight symptomatic plants from the following genera: Encyclia, Oncidium, Shomburghia, Brassia, Guarianthe, Cattleya, Epidendrum, Vanilla, Xilobium, Laelia, and Brassocattleya. Samples were tested using double-antibody sandwich (DAS)-ELISA (Agdia, Elkhart, IN) with antiserum for Cymbidium mosaic virus (CymMV), Odontoglossum ringspot virus (ORSV), Cymbidium ringspot mosaic virus, and Tobacco mosaic virus (TMV) and a general antiserum for potyviruses. At least one plant from each genus was positive to CymMV and ORSV as individual or mixed infections. Encyclia and Laelia plants were the most frequently found with mixed infections by both viruses. All genera were negative for TMV and potyviruses. Total RNA extracts were obtained from all ELISA-positive samples by a modified silica capture protocol (2). Reverse transcription (RT)-PCR was carried out with general polymerase (RdRp) gene primers corresponding to the Potexvirus group (3) and specific primers for the coat protein gene (CP) of CymMV and ORSV (1). The PCR amplification from a positive sample of each genus was resolved in agarose gels. Amplification products of the expected size were obtained for CymMV and ORSV. Five CymMV RdRp gene clones from five different plants of Laelia (GenBank Accession Nos. HQ393958, HQ393959, HQ393960, HQ393961, and HQ393962), two CP gene clones of CP gene of CymMV from two different plants of Oncidium (GenBank Accession Nos. HQ393956 and HQ393957), and three CP clones of CP of ORSV from three different plants of Encyclia (GenBank Accession Nos. HQ393953, HQ393954, and HQ393955) were sequenced. The nucleotide sequences of the Mexican orchid CymMV isolates were 96 to 97% identical to CymMV sequences in the GenBank, while those of ORSV were 99 to 100% identical to deposited ORSV sequences. To our knowledge, this is the first report of CymMV and ORSV in orchids in Mexico, which are two of the most important quarantine virus in orchids in Mexico. References: (1) P. Ajjikuttira et al. J. Gen. Virol. 86:1543, 2005. (2) J. R. Thompson et al. J. Virol. Methods 111:85, 2003. (3) R. A. A. van der Vlugt and M. Berendsen. Eur. J. Plant Pathol. 108:367, 2002.

Plant paralog to viral movement protein that potentiates transport of mRNA into the phloem.

CmPP16 from Cucurbita maxima was cloned and the protein was shown to possess properties similar to those of viral movement proteins. CmPP16 messenger RNA (mRNA) is present in phloem tissue, whereas protein appears confined to sieve elements (SE). Microinjection and grafting studies revealed that CmPP16 moves from cell to cell, mediates the transport of sense and antisense RNA, and moves together with its mRNA into the SE of scion tissue. CmPP16 possesses the characteristics that are likely required to mediate RNA delivery into the long-distance translocation stream. Thus, RNA may move within the phloem as a component of a plant information superhighway.

The phloem as a conduit for inter-organ communication.

The plant vascular system plays a pivotal role in the delivery of nutrients to distantly located organs. Recent discoveries have provided new insight into a novel role for plasmodesmata and the phloem in terms of the transport and delivery of information macromolecules (i.e. proteins and ribonucleoprotein complexes). Non-cell/organ-autonomous control over gene expression may function both in defense signaling and developmental programming in plants.

Dataset of Arabidopsis plants that overexpress FT driven by a meristem-specific KNAT1 promoter.

In this dataset we integrated figures comparing leaf number and rosette diameter in three Arabidopsis FT overexpressor lines (AtFTOE) driven by KNAT1 promoter, "A member of the KNOTTED class of homeodomain proteins encoded by the STM gene of Arabidopsis" [5], vs Wild Type (WT) Arabidopsis plats. Also, presented in the tables are some transcriptomic data obtained by RNA-seq Illumina HiSeq from rosette leaves of Arabidopsis plants of AtFTOE 2.1 line vs WT with accession numbers SRR2094583 and SRR2094587 for AtFTOE replicates 1-3 and AtWT for control replicates 1-2 respectively. Raw data of paired-end sequences are located in the public repository of the National Center for Biotechnology Information of the National Library of Medicine, National Institutes of Health, United States of America, Bethesda, MD, USA as Sequence Read Archive (SRA). Performed analyses of differential expression genes are visualized by Mapman and presented in figures. "Transcriptomic analysis of Arabidopsis overexpressing flowering locus T driven by a meristem-specific promoter that induces early flowering" [2], described the interpretation and discussion of the obtained data.

Transcriptomic analysis of Arabidopsis overexpressing flowering locus T driven by a meristem-specific promoter that induces early flowering.

Here we analyzed in leaves the effect of FT overexpression driven by meristem-specific KNAT1 gene homolog of Arabidopsis thaliana (Lincoln et al., 1994; Long et al., 1996) on the transcriptomic response during plant development. Our results demonstrated that meristematic FT overexpression generates a phenotype with an early flowering independent of photoperiod when compared with wild type (WT) plants. Arabidopsis FT-overexpressor lines (AtFTOE) did not show significant differences compared with WT lines neither in leaf number nor in rosette diameter up to day 21, when AtFTOE flowered. After this period AtFTOE plants started flower production and no new rosette leaves were produced. Additionally, WT plants continued on vegetative stage up to day 40, producing 12-14 rosette leaves before flowering. Transcriptomic analysis of rosette leaves studied by sequencing Illumina RNA-seq allowed us to determine the differential expression in mature leaf rosette of 3652 genes, being 626 of them up-regulated and 3026 down-regulated. Overexpressed genes related with flowering showed up-regulated transcription factors such as MADS-box that are known as flowering markers in meristem and which overexpression has been related with meristem identity preservation and the transition from vegetative to floral stage. Genes related with sugar transport have shown a higher demand of monosaccharides derived from the hydrolysis of sucrose to glucose and probably fructose, which can also be influenced by reproductive stage of AtFTOE plants.

Selective inhibition of cytosine-DNA methylases by polyamines.

We have advanced the hypothesis that polyamines affect DNA methylation and thus promote the expression of developmentally controlled genes. We demonstrate that the activity of cytosine-DNA methyltransferases HpaII, HhaI, HaeIII and SssI is inhibited by physiological concentrations of polyamines. On the other hand, activity of the adenine-DNA methyltransferase EcoRI, and restriction enzymes HpaII, HhaI, HaeIII and EcoRI, is insensitive to polyamine concentrations up to 40 mM. Our results indicate that the effect of polyamines on cytosine-DNA methyltransferases is rather selective and suggest a possible mode of action in vivo.

Proteolytic processing of CmPP36, a protein from the cytochrome b(5) reductase family, is required for entry into the phloem translocation pathway.

Cucurbita maxima (pumpkin) phloem sap contains a 31 kDa protein that cross-reacts with antibodies directed against the red clover necrotic mosaic virus movement protein (RCNMV MP). Microsequence data from phloem-purified 31 kDa protein were used to isolate a complementary DNA: the open reading frame encodes a 36 kDa protein belonging to the cytochrome b(5) reductase (Cb5R) family; the gene was termed CmPP36. Western analyses established that CmPP36, RCNMV MP and CmPP16 (Xoconostle-Cázares et al., 1999, Science 283, 94-98) are immunologically related, probably due to a common epitope, represented by the NADH(+)-binding domain of CmPP36. An N-terminal 5 kDa membrane-targeting domain is cleaved to produce the 31 kDa Delta N-CmPP36 detected in the phloem sap. Microinjection experiments established that Delta N-CmPP36, but not CmPP36, is able to interact with plasmodesmata to mediate its cell-to-cell transport. Thus, intercellular movement of CmPP36 requires proteolytic processing in the companion cell to produce a soluble, movement-competent, protein. In contrast to RCNMV and CmPP16, Delta N-CmPP36 interacts with but does not mediate the trafficking of RNA. Northern and in situ RT-PCR studies established that CmPP36 mRNA is present in all plant organs, being highly abundant within vascular tissues. In roots of hydroponically grown pumpkin plants, CmPP36 mRNA levels respond to changes in available iron in the culture solution. Finally, enzymatic assays established that both CmPP36 and Delta N-CmPP36 could reduce Fe(3+)-citrate and Fe(3+)-EDTA in the presence of NADH(+). These findings are discussed in terms of the possible roles played by CmPP36 in phloem function.

Phloem long-distance transport of CmNACP mRNA: implications for supracellular regulation in plants.

Direct support for the concept that RNA molecules circulate throughout the plant, via the phloem, is provided through the characterisation of mRNA from phloem sap of mature pumpkin (Cucurbita maxima) leaves and stems. One of these mRNAs, CmNACP, is a member of the NAC domain gene family, some of whose members have been shown to be involved in apical meristem development. In situ RT-PCR analysis revealed the presence of CmNACP RNA in the companion cell-sieve element complex of leaf, stem and root phloem. Longitudinal and transverse sections showed continuity of transcript distribution between meristems and sieve elements of the protophloem, suggesting CmNACP mRNA transport over long distances and accumulation in vegetative, root and floral meristems. In situ hybridization studies conducted on CmNACP confirmed the results obtained using in situ RT-PCR. Phloem transport of CmNACP mRNA was proved directly by heterograft studies between pumpkin and cucumber plants, in which CmNACP transcripts were shown to accumulate in cucumber scion phloem and apical tissues. Similar experiments were conducted with 7 additional phloem-related transcripts. Collectively, these studies established the existence of a system for the delivery of specific mRNA transcripts from the body of the plant to the shoot apex. These findings provide insight into the presence of a novel mechanism likely used by higher plants to integrate developmental and physiological processes on a whole-plant basis.

An iteron-related domain is associated to Motif 1 in the replication proteins of geminiviruses: identification of potential interacting amino acid-base pairs by a comparative approach.

Geminiviruses encode a replication initiator protein, Rep, which binds in a sequence-specific fashion to iterated DNA motifs (iterons) functioning as essential elements for virus-specific replication. By using the iterons of more than one hundred geminiviruses as heuristic devices, we have identified a Rep subdomain 8 to 10 residues in length, whose primary structure varies among viruses harboring different iterons, but which is similar among viruses with identical iterons, regardless of their differences in host range, insect vector, geographical origin or genome structure. Close analysis of this iteron-related domain (IRD) revealed consistent correlations between specific Rep residues and defined nucleotides of its cognate iteron, thus providing important insights about the molecular code which dictates the Rep preference for specific DNA sequences. A model of potential Rep-iteron contacts is proposed. The identified IRD is adjacent to a conserved motif characteristic of a superfamily of rolling-circle (RC) replication proteins, and secondary structure predictions suggest that those Rep subdomains form together the core of a novel DNA-binding domain possessing a beta-sheet as recognition subdomain, which is apparently conserved in the replication proteins of nanoviruses, circoviruses, microviruses, and a variety of ssDNA plasmids of eubacteria, archaebacteria and red algae. The evolutionary implications of these findings are discussed.

Nucleotide sequence of an osmotin-like cDNA induced in tomato during viroid infection.

A cDNA library from tomato planta macho viroid (TPMV)-infected tomato was constructed. The library was screened at low stringency with a tobacco PR-R cDNA probe. An 832 bp cDNA from a mRNA present only in infected tissue was isolated. Nucleotide sequence showed high homology with the osmotin from both tobacco and tomato (NP24). This cDNA probably corresponds to the AP24 and P23 proteins previously described in tomato and induced upon fungal and viroid infection.

Identification of a sequence element involved in AC2-mediated transactivation of the pepper huasteco virus coat protein gene.

The geminivirus AC2 gene product transactivates the expression of the coat and movement protein (CP and BV1) genes, and this effect seems to be mediated by specific although hitherto unknown cis-acting elements. In this work we examined regions from the CP and BV1 gene promoters of pepper huasteco virus (PHV) to define the sequence elements involved in regulation by AC2. Results from transient gene expression and transgenic plant assays suggest that a truncated 115-nt CP promoter is still responsive to the viral transactivator. This promoter contains three elements similar to a sequence motif termed conserved late element (CLE), which is found in the regulatory regions of many geminiviruses and that was previously suggested, on a theoretical basis, to be a potential functional target for AC2 (Argüello-Astorga et al. (1994), Virology 203, 90-100). To confirm these results, an oligonucleotide containing two CLE motifs was synthesized and characterized in gain-of-function experiments. Transient expression assays showed that this 29-nt sequence is able to confer AC2 responsiveness to heterologous promoters. A smaller oligonucleotide (16 nt) containing a single CLE also conferred this activity. In addition, when the CLE motifs were mutated in their original context (truncated 115-nt promoter), this modified promoter lost its ability to be transactivated by AC2. All these results support the involvement, at least in the case of PHV, of CLE sequences in the process of transactivation.