Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress

Giardini Naxos, Italy - 18/21 September, 2002

ISBN 88-900622-3-1

Poster Abstract - 1.20

ISOLATION OF TWO CLASSES OF RESISTANCE GENE ANALOGS IN GRAPE: NBS-LRRs AND Pto-LIKE SERINE/THREONINE KINASES

DI GASPERO G., CIPRIANI G.

Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine, Italy

grapevine, Vitis, disease, resistance, RGAs

Plant R-genes underlie a mechanism of vertical resistance in several crops, where the proteins encoded by these genes likely bind an avirulence gene product of the pathogen, thus turning on a cascade of signal transduction that activates the plant defence machinery. R-genes fall into 4 classes based on structural features of the encoded proteins: (1) cytoplasmatic serine/threonine kinases (STK) such as tomato Pto, (2) cytoplasmatic proteins with a nucleotide binding site (NBS) and a leucine-rich repeat (LRR) such as tobacco N or Arabidopsis RPS2, (3) proteins that have an extracellular LRR and a membrane-spanning domain such as tomato Cf, and (4) proteins having an extracellular LRR and a intracellular serine/threonine kinase such as rice Xa21.

NBS- and STK-domains are present in the largest three of four classes of resistance proteins, thus accounting for the majority of R-genes currently known. These domains have some functional regions whose amino acid sequence is well-conserved across several species and this make it possible to isolate resistance gene analogs (RGAs) in different crops by means of degenerate primers and PCR.

We used this candidate gene approach for the isolation of NBS- and STK-containing genes in grape using as source species Vitis amurensis, an Asian species, and Vitis riparia, a North American species, each of them carrying a putatively indipendent genetic system of resistance to downy mildew (Plasmopara viticola) together with resistances to other diseases. Downy mildew still remains the most devastating fungal disease for grapevine wherever a cool and rainy weather takes place in the early growing season.

As expected for such multi-gene families, that in Arabidopsis have some hundreds members, a pool of different fragments were cloned in grape, each one carrying the structural features of NBS- or STK-containing R-genes and showing a less extent of sequence similarity outside the putative functional domains.

Cloned NBS-containing fragments were digested with ten endonucleases and 29 out of the 71 putative recombinant clones that showed unique restriction patterns were sequenced. Using a threshold value of 40% identity, at least twelve grape NBS-sequences showed a high overall similarity with known R-genes, with the highest identity (51% and 46%) towards the Arabidopsis RPS5 and the tobacco N genes, two NBS-LRR R-genes responsible for the resistance to Peronospora parasitica and the tobacco mosaic virus, respectively. Cloned STK-fragments were analysed by SSCP and 22 out of 96 clones showing a unique sequence conformational pattern were sequenced. Nineteen grape STK-sequences contained the internal conserved motifs of other R-gene serine/threonine kinases and they showed an amino acid sequence identity up to 75% with other plant Pto-like serine/theronine kinases.

A cluster analysis of grape RGAs together with analogous domains of known R-genes, classified grape sequences into three NBS-groups and five STK-groups. Grape sequences representative of each group are under screening in either RFLP or SSCP analysis of genomic DNA from resistant and susceptible grapevine cultivars. The resistant germplasm included both wild genotypes of Vitis species used in the past decades as raw material for breeding complex hybrids and commercial cultivars, derived from those breeding programs, such as 'Seyval', 'Bianca' and 'Regent'. Up to now one NBS-sequence showed a clear-cut polymorphism between resistant and susceptible genotypes when used as a probe on Southern blots, thus representing a putative marker for resistance gene/s in Vitis.