Inhibition of crown gall induction by Agrobacterium vitis strain F2/5 in grapevine and Ricinus

Biological control measures to prevent or reduce Agrobacterium vitis-caused losses in grapevine cultures are a worldwide increasing challenge. In the present study, tumour development in grapevine (Vitis vinifera L.) was induced in the sensitive cv. Kerner by infection with Agrobacterium vitis strain K306, carrying the p35Sgus-int plasmid with the gus gene as marker for transformation by the wild-type T-DNA. Pre-inoculation with the non-tumorigenic A. vitis strain F2/5 prevented tumour induction by K306(p35gus-int). Strain M1154, a Tn5 mutant of F2/5 in the luxR-like aviR gene, partially reduced the biocontrol efficiency compared to the wild-type F2/5. GUS-labelling by K306gus was poor in grapevine in contrast to A. tumefaciens 281(p35gus-int)-induced tumours in Arabidopsis, indicating plant species-dependent variable gus expression. To use the more reliable direct mRNA expression assay by RTPCR, a new experimental plant/A. vitis system was established with Ricinus communis as model plant. Ricinus/A. vitis galls were available within one week after K306gus inoculation, reached diameters up to 5 cm, and contained more abundant GUS staining. An additional transformation marker, mRNA expression of the T-DNA-located iaaM oncogene, coding auxin synthesis, was apparent only in tumours induced by the wild-type A. vitis strain K306 in the absence of the gus construct, which is under the control of the strong 35S CaMV promoter. F2/5 pre-inoculation suppressed GUS staining and gus mRNA expression. DAPI staining revealed the loss of vital fluorescent cell nuclei in F2/5-inoculated grapevine tissue and thus inhibition of any successful T-DNA transfer into host cell nuclei. Differentiation of typical circular vessels in globular vascular bundles in M1154-pretreated galls suggests interference with plant auxin metabolism. In conclusion, together with successfully establishing a new experimental model system, Ricinus/A. vitis, pre-treatment of host tissue with the non-pathogenic strain F2/5 resulted in preventing the integration and expression of the oncogenic T-DNA of A. vitis strains by locally necrotizing host cell nuclei.

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. 'Richter 110' rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twentyone transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58

Genome of the red alga Porphyridium purpureum

The limited knowledge we have about red algal genomes comes from the highly specialized extremophiles, Cyanidiophyceae. Here, we describe the first genome sequence from a mesophilic, unicellular red alga, Porphyridium purpureum. The 8,355 predicted genes in P. purpureum, hundreds of which are likely to be implicated in a history of horizontal gene transfer, reside in a genome of 19.7 Mbp with 235 spliceosomal introns. Analysis of light-harvesting complex proteins reveals a nuclear-encoded phycobiliprotein in the alga. We uncover a complex set of carbohydrate-active enzymes, identify the genes required for the methylerythritol phosphate pathway of isoprenoid biosynthesis, and find evidence of sexual reproduction. Analysis of the compact, function-rich genome of P. purpureum suggests that ancestral lineages of red algae acted as mediators of horizontal gene transfer between prokaryotes and photosynthetic eukaryotes, thereby significantly enriching genomes across the tree of photosynthetic life

Genome, Functional Gene Annotation, and Nuclear Transformation of the Heterokont Oleaginous Alga \u3ci\u3eNannochloropsis oceanica\u3c/i\u3e CCMP1779

Unicellular marine algae have promise for providing sustainable and scalable biofuel feedstocks, although no single species has emerged as a preferred organism. Moreover, adequate molecular and genetic resources prerequisite for the rational engineering of marine algal feedstocks are lacking for most candidate species. Heterokonts of the genus Nannochloropsis naturally have high cellular oil content and are already in use for industrial production of high-value lipid products. First success in applying reverse genetics by targeted gene replacement makes Nannochloropsis oceanica an attractive model to investigate the cell and molecular biology and biochemistry of this fascinating organism group. Here we present the assembly of the 28.7 Mb genome of N. oceanica CCMP1779. RNA sequencing data from nitrogen-replete and nitrogendepleted growth conditions support a total of 11,973 genes, of which in addition to automatic annotation some were manually inspected to predict the biochemical repertoire for this organism. Among others, more than 100 genes putatively related to lipid metabolism, 114 predicted transcription factors, and 109 transcriptional regulators were annotated. Comparison of the N. oceanica CCMP1779 gene repertoire with the recently published N. gaditana genome identified 2,649 genes likely specific to N. oceanica CCMP1779. Many of these N. oceanica–specific genes have putative orthologs in other species or are supported by transcriptional evidence. However, because similarity-based annotations are limited, functions of most of these species-specific genes remain unknown. Aside from the genome sequence and its analysis, protocols for the transformation of N. oceanica CCMP1779 are provided. The availability of genomic and transcriptomic data for Nannochloropsis oceanica CCMP1779, along with efficient transformation protocols, provides a blueprint for future detailed gene functional analysis and genetic engineering of Nannochloropsis species by a growing academic community focused on this genus

Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis\textit{Porphyra umbilicalis} (Bangiophyceae, Rhodophyta)

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.The work conducted by the US Department of Energy (DOE) Joint Genome Institute, a DOE Office of Science User Facility, was supported by the Office of Science of the US DOE under Contract DE-AC02-05CH11231 (to S.H.B., E.G., A.R.G., and J.W.S.). Other major research support was provided by NSF 0929558 (to S.H.B. and A.R.G.); National Oceanic and Atmospheric Administration (NOAA) Contract NA060AR4170108 (to S.H.B.); German Research Foundation Grant Mi373/12-2 of FOR1261 (to M.M.); the French National Research Agency under IDEALG Grants ANR-10- BTBR-04-02 and 04-04 “Investissements d’avenir, Biotechnologies-Bioressources” (to J.C., E.F.-B., G.M., and S.M.D.); the New Hampshire Agricultural Experiment Station, Scientific Contribution No. 2694, supported by the US Department of Agriculture/National Institute of Food and Agriculture Hatch Project 1004051 (to A.S.K. and Y.C.); the Biotechnology and Biological Sciences Research Council (BBSRC BB/1013164/1) of the United Kingdom and European Union FP7 Marie Curie ITN Photo.Comm 317184 (to A.G.S. and K.E.H.); the Office of Biological and Environmental Research of the US DOE (C.E.B.-H.); the Connecticut Sea Grant College Program (R/A-38) and the NOAA National Marine Aquaculture Initiative (C.Y.); the NIH MCB 1244593 (to H.V.G.); NSF and NIH Grants NSF-MCB 1412738, NIH P20GM103418, and NIH P20GM103638 (to B.J.S.C.O.); NSF Graduate Research Fellowship under Grant 1247393 (to B.N.S.); the UK Natural Environment Research Council IOF Pump-priming + scheme Grant NE/L013223/1 (to C.M.M.G. and Y.B.); NOAA Contract NA14OAR4170072 (to S.H.B.); and The Great Barrier Reef Foundation, Australian Research Council (DP150101875) and a University of Queensland Early Career Researcher Grant (to C.X.C.)