Said Mouzeyar

Cloning of molecular markers for disease resistance in sunflower, Helianthus annuus L.

A candidate-gene approach to analyse the resistance of plants to phytopathogenic fungi is presented. The resistance of sunflower (Helianthus annuus L.) to downy mildew (Plasmopara halstedii) shows a gene-for-gene interaction (monogenic resistance), whereas resistance to white rot (Sclerotinia sclerotiorum) is quantitative, with different levels of resistance for different plant parts. By homology cloning, probes were obtained homologous to some plant resistance genes (nucleotide binding site-like, NBS, genes and serine-threonine protein kinase-like, PK, genes). These clones were used as probes for linkage mapping of the corresponding genes. It was demonstrated that at least three NBS-like loci are located on linkage-group 1, in the region where downy mildew resistance loci have been described. Quantitative trait loci for S. sclerotiorum resistance to penetration or extension of the mycelium in different tissues were studied in three crosses. Major QTLs for resistance were found on linkage group 1, with up to 50% of the phenotypic variability explained by peaks at the map position of the PK locus, 25 cM from the downy mildew loci.

Identification of non-TIR-NBS-LRR markers linked to the Pl5/Pl8 locus for resistance to downy mildew in sunflower

Abstract The resistance of sunflower, Helianthus annuus L., to downy mildew, caused by Plasmopara halstedii, is conferred by major genes denoted by Pl. Using degenerate and specific primers, 16 different resistance gene analogs (RGAs) have been cloned and sequenced. Sequence comparison and Southern-blot analysis distinguished six classes of RGA. Two of these classes correspond to TIR-NBS-LRR sequences while the remaining four classes correspond to the non-TIR-NBS-LRR type of resistance genes. The genetic mapping of these RGAs on two segregating F2 populations showed that the non-TIR-NBS-LRR RGAs are clustered and linked to the Pl5/Pl8 locus for resistance to downy mildew in sunflower. These and other results indicate that different Pl loci conferring resistance to the same pathogen races may contain different sequences.

Molecular analysis of a major locus for resistance to downy mildew in sunflower with specific PCR-based markers.

Abstract Resistance of sunflower to the obligate parasite Plasmopara halstedii is conferred by specific dominant genes, denoted Pl. The Pl6 locus confers resistance to all races of P. halstedii except one, and must contain at least 11 tightly linkedgenes each giving resistance to different downy mildew races. Specific primers were designed and used to amplify 13 markers covering a genetic distance of about 3 cM centred on the Pl6 locus. Cloning and sequence analysis of these 13 markers indicate that Pl6 contains conserved genes belonging to the TIR-NBS-LRR class of plant resistance genes.

Identification of a second linkage group carrying genes controlling resistance to downy mildew (Plasmopara halstedii) in sunflower (Helianthus annuus L.)

Abstract  A sunflower line, XRQ, carrying the gene Pl5, which gives resistance to all French downy mildew races shows cotyledon-limited sporulation in seedling immersion tests; consequently, segregations in crosses with other downy mildew resistance sources were tested both by this method and by a secondary infection on leaves. Pl5 was found to segregate independently of Pl7 (HA338) but to be closely linked, or allelic, with Pl8 (RHA340). F3 and F4 progenies from a cross with a line containing Pl2 showed that Pl5 carries resistance to race 100 which segregates independently of Pl2. The Pl5 gene was mapped on linkage group 6 of the Cartisol RFLP map, linked to two RFLP markers, ten AFLP markers and the restorer gene Rf1. Tests with downy mildew race 330 distinguished Pl5 and Pl8, the first being susceptible, the second resistant, whereas both these genes were active against race 304 to which Pl6 (HA335) and Pl7 gave susceptibility. It is concluded that Pl5 and Pl8 are closely linked on linkage group 6 and form a separate resistance gene group from Pl6/Pl7 on linkage group 1. The origins of these groups of downy mildew resistance genes and their use in breeding are discussed.

Development of PCR markers for the Pl5/Pl8 locus for resistance to Plasmopara halstedii in sunflower, Helianthus annuus L. from complete CC-NBS-LRR sequences

Sunflower downy mildew, caused by Plasmopara halstedii, is one of the major diseases of this crop. Development of elite sunflower lines resistant to different races of this oomycete seems to be the most efficient method to limit downy mildew damage. At least two different gene clusters conferring resistance to different races of P. halstedii have been described. In this work we report the cloning and mapping of two full-length resistance gene analogs (RGA) belonging to the CC-NBC-LRR class of plant resistance genes. The two sequences were then used to develop 14 sequence tagged sites (STS) within the Pl5/Pl8 locus conferring resistance to a wide range of P. halstedii races. These STSs will be useful in marker-assisted selection programs.

Colocation of downy mildew (Plasmopara halstedii) resistance genes in sunflower (Helianthus annuus L.)

SummaryThe Pl6 locus in the inbred sunflower (Helianthus annuus L.) line HA335 giving resistance to French races of downy mildew (Plasmopara halstedii (Farl.) Berl. & de Toni. was localized by molecular techniques. A bulked segregant analysis was made on the F2 progeny from a cross between this line and H52, a downy mildew susceptible line. The resistance gene in HA335 was found to have the same linked RFLP marker loci as those determined for Pl1 (resistance to race 1 in the line RHA266) on linkage group 1 of the consensus RFLP map of the cultivated sunflower. Pl1 and Pl6 thus appear either to be allelic or closely linked. The implications for sunflower breeding are discussed.

Down-regulation of the TaGW2 gene by RNA interference results in decreased grain size and weight in wheat

For important food crops such as wheat and rice, grain yield depends on grain number and size. In rice (Oryza sativa), GW2 was isolated from a major quantitative trait locus for yield and encodes an E3 RING ligase that negatively regulates grain size. Wheat (Triticum aestivum) has TaGW2 homologues in the A, B, and D genomes, and polymorphisms in TaGW2-A were associated with grain width. Here, to investigate TaGW2 function, RNA interference (RNAi) was used to down-regulate TaGW2 transcript levels. Transgenic wheat lines showed significantly decreased grain size-related dimensions compared with controls. Furthermore, TaGW2 knockdown also caused a significant reduction in endosperm cell number. These results indicate that TaGW2 regulates grain size in wheat, possibly by controlling endosperm cell number. Wheat and rice GW2 genes thus seem to have divergent functions, with rice GW2 negatively regulating grain size and TaGW2 positively regulating grain size. Analysis of transcription of TaGW2 homoeologues in developing grains suggested that TaGW2-A and -D act in both the division and late grain-filling phases. Furthermore, biochemical and molecular analyses revealed that TaGW2-A is a functional E3 RING ubiquitin ligase with nucleocytoplasmic subcellular partitioning. A functional nuclear export sequence responsible for TaGW2-A export from the nucleus to the cytosol and retention in the nucleolus was identified. Therefore, these results show that TaGW2 acts in the regulation of grain size and may provide an important tool for enhancement of grain yield.

Accumulation of defense related transcripts in sunflower hypocotyls (Helianthus annuus L.) infected with Plasmopara halstedii

A cDNA clone encoding a sunflower chitinase was obtained using degenerated primers in PCR amplifications and RACE procedures. This clone, a phenylalanine ammonia-lyase (PAL) clone and ubiquitin clone were used to analyse the resistance of sunflower (Helianthus annuus) to downy mildew. The differential regulation of amounts of PAL (involved in the general pathway of phenylpropanoid synthesis), chitinase (a pathogenesis-related protein) and ubiquitin (involved in proteolytic pathways) mRNA was studied in hypocotyls during the early stages after an aerial infection of sunflower inbred line RHA274 with zoospores from either race 1 (incompatible, host resistant) or race B (compatible, host susceptible) of Plasmopara halstedii. Northern analyses showed that transcript levels of PAL, chitinase and ubiquitin were rapidly and strongly increased after infection in incompatible interactions but not in the compatible ones, suggesting that regulation of these mRNAs is an important component of the resistance mechanisms in sunflower.

Biochemical identification of proteasome-associated endonuclease activity in sunflower

Proteasomes have been purified from sunflower hypocotyles. They elute with a molecular mass of 600 kDa from gel filtration columns and two-dimensional gel electrophoresis indicates that the complex contains at least 20 different protein subunits. Peptide microsequencing revealed the presence of four subunits homologous to subunits Beta2, Beta6, Alpha5 and Alpha6 of plant proteasomes. These proteasomes have chymotrypsin-like activity and the highly purified fraction of this complex is associated with an endonuclease activity hydrolyzing Tobacco mosaic virus RNA and Lettuce mosaic virus RNA with a cleavage pattern showing fragments of well-defined size. This is the first evidence of a RNA endonuclease activity associated with plant proteasomes.

Antisense expression of a NBS-LRR sequence in sunflower (Helianthus annuus L.) and tobacco (Nicotiana tabacum L.): evidence for a dual role in plant development and fungal resistance.

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.