Angelica Jermakow

Gibberellins Are Required for Seed Development and Pollen Tube Growth in Arabidopsis

Gibberellins (GAs) are tetracyclic diterpenoids that are essential endogenous regulators of plant growth and development. GA levels within the plant are regulated by a homeostatic mechanism that includes changes in the expression of a family of GA-inactivating enzymes known as GA 2-oxidases. Ectopic expression of a pea GA 2-oxidase2 cDNA caused seed abortion in Arabidopsis, extending and confirming previous observations obtained with GA-deficient mutants of pea, suggesting that GAs have an essential role in seed development. A new physiological role for GAs in pollen tube growth in vivo also has been identified. The growth of pollen tubes carrying the 35S:2ox2 transgene was reduced relative to that of nontransgenic pollen, and this phenotype could be reversed partially by GA application in vitro or by combining with spy-5, a mutation that increases GA response. Treatment of wild-type pollen tubes with an inhibitor of GA biosynthesis in vitro also suggested that GAs are required for normal pollen tube growth. These results extend the known physiological roles of GAs in Arabidopsis development and suggest that GAs are required for normal pollen tube growth, a physiological role for GAs that has not been established previously.

A 48 SNP set for grapevine cultivar identification

BackgroundRapid and consistent genotyping is an important requirement for cultivar identification in many crop species. Among them grapevine cultivars have been the subject of multiple studies given the large number of synonyms and homonyms generated during many centuries of vegetative multiplication and exchange. Simple sequence repeat (SSR) markers have been preferred until now because of their high level of polymorphism, their codominant nature and their high profile repeatability. However, the rapid application of partial or complete genome sequencing approaches is identifying thousands of single nucleotide polymorphisms (SNP) that can be very useful for such purposes. Although SNP markers are bi-allelic, and therefore not as polymorphic as microsatellites, the high number of loci that can be multiplexed and the possibilities of automation as well as their highly repeatable results under any analytical procedure make them the future markers of choice for any type of genetic identification.ResultsWe analyzed over 300 SNP in the genome of grapevine using a re-sequencing strategy in a selection of 11 genotypes. Among the identified polymorphisms, we selected 48 SNP spread across all grapevine chromosomes with allele frequencies balanced enough as to provide sufficient information content for genetic identification in grapevine allowing for good genotyping success rate. Marker stability was tested in repeated analyses of a selected group of cultivars obtained worldwide to demonstrate their usefulness in genetic identification.ConclusionsWe have selected a set of 48 stable SNP markers with a high discrimination power and a uniform genome distribution (2-3 markers/chromosome), which is proposed as a standard set for grapevine (Vitis vinifera L.) genotyping. Any previous problems derived from microsatellite allele confusion between labs or the need to run reference cultivars to identify allele sizes disappear using this type of marker. Furthermore, because SNP markers are bi-allelic, allele identification and genotype naming are extremely simple and genotypes obtained with different equipments and by different laboratories are always fully comparable.

Identification of grapevine MLO gene candidates involved in susceptibility to powdery mildew

The European cultivated grapevine, Vitis vinifera L., is a host for the powdery mildew pathogen Erisyphe necator, which is the most economically important fungal disease of viticulture. MLO proteins mediate powdery mildew susceptibility in the model plant species Arabidopsis and the crop plants barley and tomato. Seven VvMLO cDNA sequences were isolated from grapevine and were subsequently identified as part of a 17 member VvMLO gene family within the V. vinifera genome. Phylogenetic analysis of the 17 VvMLO genes in the grape genome indicated that the proteins they encode fall into six distinct clades. The expression of representative VvMLOs from each clade were analysed in a range of grape tissues, as well as in response to a range of biotic and abiotic factors. The VvMLOs investigated have unique, but overlapping tissue expression patterns. Expression analysis of VvMLO genes following E. necator infection identified four upregulated VvMLOs which are orthologous to the Arabidopsis AtMLO2, AtMLO6 and AtMLO12 and tomato SlMLO1 genes required for powdery mildew susceptibility. This suggests a degree of functional redundancy between the proteins encoded by these genes in terms of susceptibility to powdery mildew, and, as such, represent potential targets for modification to generate powdery mildew resistant grapevines.

Genetic dissection of a TIR-NB-LRR locus from the wild North American grapevine species Muscadinia rotundifolia identifies paralogous genes conferring resistance to major fungal and oomycete pathogens in cultivated grapevine.

The most economically important diseases of grapevine cultivation worldwide are caused by the fungal pathogen powdery mildew (Erysiphe necator syn. Uncinula necator) and the oomycete pathogen downy mildew (Plasmopara viticola). Currently, grapegrowers rely heavily on the use of agrochemicals to minimize the potentially devastating impact of these pathogens on grape yield and quality. The wild North American grapevine species Muscadinia rotundifolia was recognized as early as 1889 to be resistant to both powdery and downy mildew. We have now mapped resistance to these two mildew pathogens in M. rotundifolia to a single locus on chromosome 12 that contains a family of seven TIR-NB-LRR genes. We further demonstrate that two highly homologous (86% amino acid identity) members of this gene family confer strong resistance to these unrelated pathogens following genetic transformation into susceptible Vitis vinifera winegrape cultivars. These two genes, designated resistance to Uncinula necator (MrRUN1) and resistance to Plasmopara viticola (MrRPV1) are the first resistance genes to be cloned from a grapevine species. Both MrRUN1 and MrRPV1 were found to confer resistance to multiple powdery and downy mildew isolates from France, North America and Australia; however, a single powdery mildew isolate collected from the south-eastern region of North America, to which M. rotundifolia is native, was capable of breaking MrRUN1-mediated resistance. Comparisons of gene organization and coding sequences between M. rotundifolia and the cultivated grapevine V. vinifera at the MrRUN1/MrRPV1 locus revealed a high level of synteny, suggesting that the TIR-NB-LRR genes at this locus share a common ancestor.

Overexpression of a gibberellin inactivation gene alters seed development, KNOX gene expression, and plant development in Arabidopsis.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

Host Cell Entry of Powdery Mildew Is Correlated with Endosomal Transport of Antagonistically Acting VvPEN1 and VvMLO to the Papilla

Challenge by a nonadapted powdery mildew fungal pathogen leads to the formation of a local cell-wall apposition (papilla) beneath the point of attempted penetration. Several plasma membrane (PM) proteins with opposing roles in powdery mildew infection, including Arabidopsis thaliana PENETRATION1 (PEN1) and barley (Hordeum vulgare) MILDEW RESISTANCE LOCUS O (MLO), are localized to the site of powdery mildew attack. PEN1 contributes to penetration resistance to nonadapted powdery mildews, whereas MLO is a susceptibility factor required by adapted powdery mildew pathogens for host cell entry. Our previous studies have demonstrated that the vesicle and endosomal trafficking inhibitors, brefeldin A and wortmannin, have opposite effects on the penetration rates of adapted and nonadapted powdery mildews on grapevine. These findings prompted us to study the pathogen-induced intracellular trafficking of grapevine variants of MLO and PEN1. We first identified grapevine (Vitis vinifera) VvPEN1 and VvMLO orthologs that rescue Arabidopsis Atpen1 and Atmlo2 mlo6 mlo12 null mutants, respectively. By using endomembrane trafficking inhibitors in combination with fluorescence microscopy, we demonstrate that VvMLO3/VvMLO4 and VvPEN1 are co-trafficked together from the PM to the site of powdery mildew challenge. This focal accumulation of VvMLO3/VvMLO4 and VvPEN1 to the site of attack seems to be required for their opposing functions during powdery mildew attack, because their subcellular localization is correlated with the outcome of attempted powdery mildew penetration.

Grapevine MLO candidates required for powdery mildew pathogenicity

MLOs belong to the largest family of seven-transmembrane (7TM) domain proteins found in plants. The Arabidopsis and rice genomes contain 15 and 12 MLO family members, respectively. Although the biological function of most MLO family members remains elusive, a select group of MLO proteins have been demonstrated to negatively regulate defence responses to the obligate biotrophic pathogen, powdery mildew, thereby acting as “susceptibility” genes. Recently we identified a family of 17 putative VvMLO genes in the genome of the cultivated winegrape species, Vitis vinifera. Expression analysis indicated that the VvMLO family members respond differently to biotic and abiotic stimuli. Infection of V. vinifera by grape powdery mildew (Erisyphe necator) specifically up-regulates four VvMLO genes that are orthologous to the Arabidopsis and tomato MLOs previously demonstrated to be required for powdery mildew susceptibility. We postulate that one or more of these E. necator responsive VvMLOs may have a role in the powdery mildew susceptibility of grapevine.

Preliminary development of a genetic strategy to prevent transgene escape by blocking effective pollen flow from transgenic plants

Genetic modification (GM) of plants has great potential in the production of food and industrial compounds, and in molecular pharming. One of the greatest public concerns regarding this technology is effective pollen flow, in which wind- or insect-borne transgenic pollen is able to fertilise either non-GM crops of the same species, or closely related weed species, and lead to viable seed formation. In this paper we describe a novel concept, based on epigenetic inheritance (imprinting) and post-transcriptional gene silencing (PTGS)/RNA interference (RNAi), designed to prevent transgene escape via pollen flow from transgenic plants. A key advantage of this strategy is that it would allow all seeds from self-pollinated transgenic plants to be harvested and re-sown, without the need for specific treatments, while retaining all of the transgenes present in the parent. Thus, this strategy is not a Genetic Use Restriction Technology (GURT) and if implemented would not prevent seed saving by end-users.