Bovine papillomavirus 24: a novel member of the genus Xipapillomavirus detected in the Amazon region.

Bovine papillomaviruses (BPVs) have been described as etiologic agents of cutaneous and mucosal papillomas in cattle. In the present study, we describe a new BPV that was detected in a cutaneous papilloma from a cow. Phylogenetic analysis suggests that this virus belong to the genus Xipapillomavirus, and we refer to it here as BPV type 24 (BPV24). Coinfection with members of the genera Epsilonpapillomavirus and Deltapapillomavirus in a cutaneous papilloma from a different animal was also detected, and the full genomes of these viruses were sequenced. Both papillomas were from cattle within Acre State in the Amazon region of Brazil. The data presented here demonstrate the utility of using high-throughput methods to indentify coinfections and allow the characterization of new genomes.

Medicago truncatula SOC1 Genes Are Up-regulated by Environmental Cues That Promote Flowering.

Like Arabidopsis thaliana, the flowering of the legume Medicago truncatula is promoted by long day (LD) photoperiod and vernalization. However, there are differences in the molecular mechanisms involved, with orthologs of two key Arabidopsis thaliana regulators, FLOWERING LOCUS C (FLC) and CONSTANS (CO), being absent or not having a role in flowering time function in Medicago. In Arabidopsis, the MADS-box transcription factor gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (AtSOC1), plays a key role in integrating the photoperiodic and vernalization pathways. In this study, we set out to investigate whether the Medicago SOC1 genes play a role in regulating flowering time. Three Medicago SOC1 genes were identified and characterized (MtSOC1a-MtSOC1c). All three MtSOC1 genes, when heterologously expressed, were able to promote earlier flowering of the late-flowering Arabidopsis soc1-2 mutant. The three MtSOC1 genes have different patterns of expression. However, consistent with a potential role in flowering time regulation, all three MtSOC1 genes are expressed in the shoot apex and are up-regulated in the shoot apex of plants in response to LD photoperiods and vernalization. The up-regulation of MtSOC1 genes was reduced in Medicago fta1-1 mutants, indicating that they are downstream of MtFTa1. Insertion mutant alleles of Medicago soc1b do not flower late, suggestive of functional redundancy among Medicago SOC1 genes in promoting flowering.

Genomic characterisation of Felis catus papillomavirus type 5 with proposed classification within a new papillomavirus genus.

Four Felis catus papillomavirus (FcaPV) types have been fully sequenced from domestic cats. Of these, FcaPV-2 and -3 are thought to cause feline viral plaques and Bowenoid in situ carcinomas. Two short sequences of DNA from a previously unreported PV type were amplified from a feline viral plaque using consensus PCR primers. DNA was then extracted from a swab of the lesion and two sets of 'outward facing' primers were designed using the short sequences to amplify the entire 7600bp genome of the novel PV. The PV was designated FcaPV-5 and contained putative coding regions that were predicted to produce five early proteins and two late ones. The ORF L1 showed over 65% similarity to that of FcaPV-3 and -4. Assignment to a genus was difficult as the PV was over 60% similar to PV types from 4 different genera. However, due to the ORF L1 similarity of FcaPV-3, -4, and -5, the shared host species of all three PVs, and the similar lesions associated with FcaPV-3 and -5, it is proposed all three PVs are classified within a new genus. FcaPV-5 is the third PV type to be associated with feline viral plaques. The plaque that contained FcaPV-5 showed unusual histological features including hyperplasia and PV-induced cell changes in sebaceous glands and deep within hair follicles. While additional study of further lesions of this type is required, it is possible that FcaPV-5 may be able to infect a broader range of cells than other PV types.

Characterization of the cyanobacteria and associated bacterial community from an ephemeral wetland in New Zealand.

New Zealand ephemeral wetlands are ecologically important, containing up to 12% of threatened native plant species and frequently exhibiting conspicuous cyanobacterial growth. In such environments, cyanobacteria and associated heterotrophs can influence primary production and nutrient cycling. Wetland communities, including bacteria, can be altered by increased nitrate and phosphate due to agricultural practices. We have characterized cyanobacteria from the Wairepo Kettleholes Conservation Area and their associated bacteria. Use of 16S rRNA amplicon sequencing identified several operational taxonomic units (OTUs) representing filamentous heterocystous and non-heterocystous cyanobacterial taxa. One Nostoc OTU that formed macroscopic colonies dominated the cyanobacterial community. A diverse bacterial community was associated with the Nostoc colonies, including a core microbiome of 39 OTUs. Identity of the core microbiome associated with macroscopic Nostoc colonies was not changed by the addition of nutrients. One OTU was highly represented in all Nostoc colonies (27.6%-42.6% of reads) and phylogenetic analyses identified this OTU as belonging to the genus Sphingomonas. Scanning electron microscopy showed the absence of heterotrophic bacteria within the Nostoc colony but revealed a diverse community associated with the colonies on the external surface.

Genomic characterization of a novel Epsilonpapillomavirus associated with pigmented papillomas in a red deer (Cervus elaphus).

Two of a group of 15 farmed European red (Cervus elaphus elaphus) X wapiti (C. e. canadensis) deer stags developed multiple persistent pigmented squamous papillomas (warts) on their chins. DNA was extracted from a papilloma and a short section of DNA from a novel papillomavirus (PV) was amplified. This short sequence was used to design 'outward facing' primers to amplify the remainder of the circular PV DNA. The PCR product was sequenced using next-generation sequencing and the full genome of the PV, consisting of 8082 bp, was assembled and analysed. The novel PV was designated Cervus elaphus papillomavirus (CePV) type 2. The putative coding regions of CePV2 were predicted to produce four early and two late proteins with two other potential ORFs also noted. Phylogenetic analysis of ORF L1 revealed greater than 60 %, but less than 70 % similarity, to Bos taurus papillomavirus (BPV) types -5 and -7. As both BPV5 and BPV7 are Epsilonpapillomavirus 1, CePV2 is proposed as the first Epsilonpapillomavirus 2 PV type. This is the first EpsilonPV to be identified in a non-bovine species and the first non-DeltaPV to be identified as a cause of disease in any deer species.

Genomic characterisation of canine papillomavirus type 17, a possible rare cause of canine oral squamous cell carcinoma.

Squamous cell carcinomas (SCCs) are the second most common cancer of the canine oral cavity resulting in significant morbidity and mortality. Recently a dog with multiple oral SCCs that contained a novel papillomavirus (PV) was reported. The aim of the present study was to determine the genome of this novel PV. To do this a short section of PV DNA was amplified from an oral SCC and 'back-to-back' primers were designed. Due to the circular nature of PV DNA, these primers were then used to amplify the remainder of the genome by inverse PCR. The PCR product was sequenced using next generation sequencing and the full genome of the PV, consisting of 8007 bp, was assembled and analysed. As this is the seventeenth PV identified from the domestic dog, the novel PV was designated Canis familiaris papillomavirus (CPV) type 17. Similar to other CPV types, the putative coding regions of CPV-17 were predicted to produce 5 early and 2 late proteins. Phylogenetic analysis of ORF L1 revealed greater than 70% similarity to CPV-2 and CPV-7 and we propose that CPV-17 also be classified as a Taupapillomavirus 1. While it appears CPV-17 is only rarely present in canine oral SCCs, evidence suggests that this PV could influence the development of oral SCCs in this species.

Genomic characterisation of the feline sarcoid-associated papillomavirus and proposed classification as Bos taurus papillomavirus type 14.

Feline sarcoids are rare mesenchymal neoplasms of domestic and exotic cats. Previous studies have consistently detected short DNA sequences from a papillomavirus (PV), designated feline sarcoid-associated papillomavirus (FeSarPV), in these neoplasms. The FeSarPV sequence has never been detected in any non-sarcoid sample from cats but has been amplified from the skin of cattle suggesting that feline sarcoids are caused by cross-species infection by a bovine papillomavirus (BPV). The aim of the present study was to determine the genome of the PV that contains the FeSarPV sequence. Using the circular nature of PV DNA, four specifically designed 'outward facing' primers were used to amplify two approximately 4,000 bp DNA segments from a feline sarcoid. The two PCR products were sequenced using next generation sequencing and the full genome of the PV, consisting 7,966 bp, was assembled and analysed. Phylogenetic analysis revealed the PV was closely related to the species 4 delta BPVs-1, -2, and -13, but distantly related to any carnivoran PV genus. These results are consistent with feline sarcoids being caused by a BPV type and we propose a classification of BPV-14 for this novel PV. Initial analysis suggests that, like other delta BPVs, the BPV-14 E5 protein could cause mesenchymal proliferation by binding to the platelet derived growth factor beta receptor. Interestingly BPV-14 has not been detected in any equine sarcoid suggesting that BPV-14 has a host range that is limited to bovids and felids.

Isolation and functional analysis of CONSTANS-LIKE genes suggests that a central role for CONSTANS in flowering time control is not evolutionarily conserved in Medicago truncatula.

The zinc finger transcription factor CONSTANS has a well-established central role in the mechanism for photoperiod sensing in Arabidopsis, integrating light and circadian clock signals to upregulate the florigen gene FT under long-day but not short-day conditions. Although CONSTANS-LIKE (COL) genes in other species have also been shown to regulate flowering time, it is not clear how widely this central role in photoperiod sensing is conserved. Legumes are a major plant group and various legume species show significant natural variation for photoperiod responsive flowering. Orthologs of several Arabidopsis genes have been shown to participate in photoperiodic flowering in legumes, but the possible function of COL genes as integrators of the photoperiod response has not yet been examined in detail. Here we characterize the COL family in the temperate long-day legume Medicago truncatula, using expression analyses, reverse genetics, transient activation assays and Arabidopsis transformation. Our results provide several lines of evidence suggesting that COL genes are unlikely to have a central role in the photoperiod response mechanism in this species.

Genomic characterisation of Felis catus papillomavirus 4, a novel papillomavirus detected in the oral cavity of a domestic cat.

Three papillomaviruses (PVs) from the domestic cat have been fully sequenced so far including Felis domesticus PV-1 (FdPV-1), FdPV-2, and a recently described Felis catus PV-3 (FcaPV-4). In the current article, we describe the full genomic sequence of a fourth PV from the domestic cat. This PV was amplified from the oral cavity of a cat with severe gingivitis. However, the aetiological involvement of FcaPV-4 in development of lesions observed in this cat remains uncertain. The complete genome of the novel virus comprised 7,616 bp and was predicted to encode five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) genes, with the organisation typical for PVs. The L1 showed 65.1 % nucleotide sequence identity to L1 of FcaPV-3 and approximately 60 % identity to L1 of canine tau-papillomaviruses CPV-2 and CPV-7. The novel virus clustered with FcaPV-3, CPV-2 and CPV-7 on a phylogenetic tree constructed from a concatenated alignment of 3,013 bp from E1, E2, L1 and L2. Based on the genomic and phylogenetic data, we propose that the novel virus is classified as a distinct species within the same genus as FcaPV-3. We also propose that both viruses are classified within the genus Taupapillomavirus, although this classification may need to be re-visited after more tau-PV genomes become available.

Genomic characterization of Felis catus papillomavirus-3: a novel papillomavirus detected in a feline Bowenoid in situ carcinoma.

There is increasing evidence that papillomaviruses (PVs) may cause skin cancer in cats. Neoplasms most frequently contain Felis domesticus PV type 2 (FdPV-2) DNA, but other PV DNA sequences have also been detected suggesting multiple PVs could cause disease. One of these sequences, FdPV-MY2, was previously detected in 5 of a series of 70 feline skin cancers. The aim was to determine the genome sequence of this PV. Using the circular nature of PV DNA, 'outward facing' primers specific for FdPV-MY2 were designed and amplified a 7300 bp length of DNA from a feline Bowenoid in situ carcinoma (BISC) that showed microscopic evidence of a viral etiology and tested positive for FdPV-MY2 DNA. The PCR product was sequenced using next generation sequencing technology. The full genomic sequence of the virus, comprising 7583 bp, was assembled and analyzed. As this is the third PV from a domestic cat, the virus was designated Felis catus PV type 3 (FcaPV-3). Consistent with other PVs, the putative coding regions of FcaPV-3 were predicted to produce 6 early proteins and 2 late ones. Classification was difficult as the virus contained over 60% nucleotide similarity within the ORF L1 with PVs from 3 different genera. However, based on phylogenetic analysis of ORF L1, FcaPV-3 was most closely related to the tau-PVs CPV-2 and CPV-7. As FcaPV-3 has over 60% nucleotide similarity with the ORF L1 of both tau-PVs, it is proposed that FcaPV-3 is classified in the genus Taupapillomavirus and is the first non-canine PV in this genus.

A conserved molecular basis for photoperiod adaptation in two temperate legumes.

Legumes were among the first plant species to be domesticated, and accompanied cereals in expansion of agriculture from the Fertile Crescent into diverse environments across the Mediterranean basin, Europe, Central Asia, and the Indian subcontinent. Although several recent studies have outlined the molecular basis for domestication and eco-geographic adaptation in the two main cereals from this region, wheat and barley, similar questions remain largely unexplored in their legume counterparts. Here we identify two major loci controlling differences in photoperiod response between wild and domesticated pea, and show that one of these, high response to photoperiod (HR), is an ortholog of early flowering 3 (ELF3), a gene involved in circadian clock function. We found that a significant proportion of flowering time variation in global pea germplasm is controlled by HR, with a single, widespread functional variant conferring altered circadian rhythms and the reduced photoperiod response associated with the spring habit. We also present evidence that ELF3 has a similar role in lentil, another major legume crop, with a distinct functional variant contributing to reduced photoperiod response in cultivars widely deployed in short-season environments. Our results identify the factor likely to have permitted the successful prehistoric expansion of legume cultivation to Northern Europe, and define a conserved genetic basis for major adaptive changes in flowering phenology and growth habit in an important crop group.

The Medicago FLOWERING LOCUS T homolog, MtFTa1, is a key regulator of flowering time.

FLOWERING LOCUS T (FT) genes encode proteins that function as the mobile floral signal, florigen. In this study, we characterized five FT-like genes from the model legume, Medicago (Medicago truncatula). The different FT genes showed distinct patterns of expression and responses to environmental cues. Three of the FT genes (MtFTa1, MtFTb1, and MtFTc) were able to complement the Arabidopsis (Arabidopsis thaliana) ft-1 mutant, suggesting that they are capable of functioning as florigen. MtFTa1 is the only one of the FT genes that is up-regulated by both long days (LDs) and vernalization, conditions that promote Medicago flowering, and transgenic Medicago plants overexpressing the MtFTa1 gene flowered very rapidly. The key role MtFTa1 plays in regulating flowering was demonstrated by the identification of fta1 mutants that flowered significantly later in all conditions examined. fta1 mutants do not respond to vernalization but are still responsive to LDs, indicating that the induction of flowering by prolonged cold acts solely through MtFTa1, whereas photoperiodic induction of flowering involves other genes, possibly MtFTb1, which is only expressed in leaves under LD conditions and therefore might contribute to the photoperiodic regulation of flowering. The role of the MtFTc gene is unclear, as the ftc mutants did not have any obvious flowering-time or other phenotypes. Overall, this work reveals the diversity of the regulation and function of the Medicago FT family.

The pea GIGAS gene is a FLOWERING LOCUS T homolog necessary for graft-transmissible specification of flowering but not for responsiveness to photoperiod.

Garden pea (Pisum sativum) was prominent in early studies investigating the genetic control of flowering and the role of mobile flowering signals. In view of recent evidence that genes in the FLOWERING LOCUS T (FT) family play an important role in generating mobile flowering signals, we isolated the FT gene family in pea and examined the regulation and function of its members. Comparison with Medicago truncatula and soybean (Glycine max) provides evidence of three ancient subclades (FTa, FTb, and FTc) likely to be common to most crop and model legumes. Pea FT genes show distinctly different expression patterns with respect to developmental timing, tissue specificity, and response to photoperiod and differ in their activity in transgenic Arabidopsis thaliana, suggesting they may have different functions. We show that the pea FTa1 gene corresponds to the GIGAS locus, which is essential for flowering under long-day conditions and promotes flowering under short-day conditions but is not required for photoperiod responsiveness. Grafting, expression, and double mutant analyses show that GIGAS/FTa1 regulates a mobile flowering stimulus but also provide clear evidence for a second mobile flowering stimulus that is correlated with expression of FTb2 in leaf tissue. These results suggest that induction of flowering by photoperiod in pea results from interactions among several members of a diversified FT family.

Noncanonical translation initiation of the Arabidopsis flowering time and alternative polyadenylation regulator FCA.

The RNA binding protein FCA regulates the floral transition and is required for silencing RNAs corresponding to specific noncoding sequences in the Arabidopsis thaliana genome. Through interaction with the canonical RNA 3' processing machinery, FCA affects alternative polyadenylation of many transcripts, including antisense RNAs at the locus encoding the floral repressor FLC. This potential for widespread alteration of gene regulation clearly needs to be tightly regulated, and we have previously shown that FCA expression is autoregulated through poly(A) site choice. Here, we show distinct layers of FCA regulation that involve sequences within the 5' region that regulate noncanonical translation initiation and alter the expression profile. FCA translation in vivo occurs exclusively at a noncanonical CUG codon upstream of the first in-frame AUG. We fully define the upstream flanking sequences essential for its selection, revealing features that distinguish this from other non-AUG start site mechanisms. Bioinformatic analysis identified 10 additional Arabidopsis genes that likely initiate translation at a CUG codon. Our findings reveal further unexpected complexity in the regulation of FCA expression with implications for its roles in regulating flowering time and gene expression and more generally show plant mRNA exceptions to AUG translation initiation.

FRIGIDA and related proteins have a conserved central domain and family specific N- and C- terminal regions that are functionally important.

Arabidopsis accessions are either winter-annuals, which require cold winter temperatures for spring flowering, or rapid-cycling summer annuals. Typically, winter annual accessions have functional FRIGIDA (FRI) and FRIGIDA-LIKE 1 (FRL1) proteins that promote high expression of FLOWERING LOCUS C (FLC), which prevents flowering until after winter. In contrast, many rapid-cycling accessions have low FLC levels because FRI is inactive. Using biochemical, functional and bioinformatic approaches, we show that FRI and FRL1 contain a stable, central domain that is conserved across the FRI superfamily. This core domain is monomeric in solution and primarily alpha-helical. We analysed the ability of several FRI deletion constructs to function in Arabidopsis plants. Our findings suggest that the C-terminus, which is predicted to be disordered, is required for FRI to promote FLC expression and may mediate protein:protein interactions. The contribution of the FRI N-terminus appears to be limited, as constructs missing these residues retained significant activity when expressed at high levels. The important N- and C-terminal regions differ between members of the FRI superfamily and sequence analysis identified five FRI families with distinct expression patterns in Arabidopsis, suggesting the families have separate biological roles.

DIE NEUTRALIS and LATE BLOOMER 1 contribute to regulation of the pea circadian clock.

The DIE NEUTRALIS (DNE) locus in garden pea (Pisum sativum) was previously shown to inhibit flowering under noninductive short-day conditions and to affect a graft-transmissible flowering signal. In this study, we establish that DNE has a role in diurnal and/or circadian regulation of several clock genes, including the pea GIGANTEA (GI) ortholog LATE BLOOMER 1 (LATE1) and orthologs of the Arabidopsis thaliana genes LATE ELONGATED HYPOCOTYL and TIMING OF CHLOROPHYLL A/B BINDING PROTEIN EXPRESSION 1. We also confirm that LATE1 participates in the clock and provide evidence that DNE is the ortholog of Arabidopsis EARLY FLOWERING4 (ELF4). Circadian rhythms of clock gene expression in wild-type plants under constant light were weaker in pea than in Arabidopsis, and a number of differences were also seen in the effects of both DNE/ELF4 and LATE1/GI on clock gene expression. Grafting studies suggest that DNE controls flowering at least in part through a LATE1-dependent mobile stimulus, and dne mutants show elevated expression of a FLOWERING LOCUS T homolog under short-day conditions. However, the early flowering of the dne mutant is not associated with altered expression of a previously described CONSTANS-like gene. Collectively, our results characterize the clock system and reveal its importance for photoperiod responsiveness in a model legume.