Inmaculada Hernández-Pinzón

Composition and role of tapetal lipid bodies in the biogenesis of the pollen coat of Brassica napus

Abstract. The composition of the two major lipidic organelles of the tapetum of Brassica napus L. has been determined. Elaioplasts contained numerous small (0.2–0.6 μm) lipid bodies that were largely made up of sterol esters and triacylglycerols, with monogalactosyldiacylglycerol as the major polar lipid. This is the first report in any species of the presence of non-cytosolic, sterol ester-rich, lipid bodies. The elaioplast lipid bodies also contained 34- and 36-kDa proteins which were shown by N-terminal sequencing to be homologous to fibrillin and other plastid lipid-associated proteins. Tapetosomes contained mainly polyunsaturated triacylglycerols and associated phospholipids plus a diverse class of oleosin-like proteins. The pollen coat, which is derived from tapetosomes and elaioplasts, was largely made up of sterol esters and the C-terminal domains of the oleosin-like proteins, but contained virtually no galactolipids, triacylglycerols or plastid lipid-associated proteins. The sterol compositions of the elaioplast and pollen coat were almost identical, consisting of stigmasterol > campestdienol > campesterol > sitosterol ≫ cholesterol, which is consistent with the majority of the pollen coat lipids being derived from elaioplasts. These data demonstrate that there is substantial remodelling of both the lipid and protein components of elaioplasts and tapetosomes following their release into the anther locule from lysed tapetal cells, and that components of both organelles contribute to the formation of the lipidic coating of mature pollen grains.

Rapid cloning of disease-resistance genes in plants using mutagenesis and sequence capture

Wild relatives of domesticated crop species harbor multiple, diverse, disease resistance (R) genes that could be used to engineer sustainable disease control. However, breeding R genes into crop lines often requires long breeding timelines of 5–15 years to break linkage between R genes and deleterious alleles (linkage drag). Further, when R genes are bred one at a time into crop lines, the protection that they confer is often overcome within a few seasons by pathogen evolution. If several cloned R genes were available, it would be possible to pyramid R genes in a crop, which might provide more durable resistance. We describe a three-step method (MutRenSeq)-that combines chemical mutagenesis with exome capture and sequencing for rapid R gene cloning. We applied MutRenSeq to clone stem rust resistance genes Sr22 and Sr45 from hexaploid bread wheat. MutRenSeq can be applied to other commercially relevant crops and their relatives, including, for example, pea, bean, barley, oat, rye, rice and maize.

The Frequent Transcriptional Readthrough of the Tobacco Tnt1 Retrotransposon and Its Possible Implications for the Control of Resistance Genes

Retrotransposons are a major component of eukaryote genomes, being especially abundant in plant genomes. They are frequently found inserted in gene-rich regions and have greatly contributed to the evolution of gene coding capacity and regulation. Retrotransposon insertions can influence the expression of neighboring genes in many ways, such as modifying their promoter or terminator sequences and altering their epigenetic control. Plant retrotransposons are highly regulated and their expression is usually associated with stress situations. While the control of transcription of some plant retrotransposons has been analyzed in some detail, little is known about the transcriptional termination of these elements. Here we show that the transcripts of the tobacco retrotransposon Tnt1 display a high variability of polyadenylation sites, only a fraction of them terminating at the major termination site. We also report on the ability of Tnt1 to extend its transcription into flanking genomic sequences and we analyze a particular case in which Tnt1 transcripts include sequences of an oppositely oriented resistance-like gene. The expression of this gene and the neighboring Tnt1 copy generate transcripts overlapping in more that 800 nucleotides, which could anneal and form dsRNAs and enter into silencing regulatory pathways. Resistance gene loci are usually composed of tandem arrays of resistance-like genes, a number of which contain mutations, including retrotransposon insertions, and are considered as to be pseudogenes. Given that plant retrotransposons are usually regulated by stress, the convergent expression of these resistance-like pseudogenes and the interleaving inducible retrotransposons may contribute to the control of plant responses to stress.

Characterisation and analysis of the Aegilops sharonensis transcriptome, a wild relative of wheat in the Sitopsis section.

Aegilops sharonensis Eig (Sharon goatgrass) is a wild diploid relative of wheat within the Sitopsis section of Aegilops . This species represents an untapped reservoir of genetic diversity for traits of agronomic importance, especially as a source of novel disease resistance. To gain a foothold in this genetic resource, we sequenced the cDNA from leaf tissue of two geographically distinct Ae . sharonensis accessions (1644 and 2232) using the 454 Life Sciences platform. We compared the results of two different assembly programs using different parameter sets to generate 13 distinct assemblies in an attempt to maximize representation of the gene space in de novo transcriptome assembly. The most sensitive assembly (71,029 contigs; N50 674 nts) retrieved 18,684 unique best reciprocal BLAST hits (BRBH) against six previously characterised grass proteomes while the most specific assembly (30,609 contigs; N50 815 nts) retrieved 15,687 BRBH. We combined these two assemblies into a set of 62,243 non-redundant sequences and identified 139 belonging to plant disease resistance genes of the nucleotide binding leucine-rich repeat class. Based on the non-redundant sequences, we predicted 37,743 single nucleotide polymorphisms (SNP), equivalent to one per 1,142 bp. We estimated the level of heterozygosity as 1.6% in accession 1644 and 30.1% in 2232. The Ae . sharonensis leaf transcriptome provides a rich source of sequence and SNPs for this wild wheat relative. These sequences can be used with existing monocot genome sequences and EST sequence collections (e.g. barley, Brachypodium , wheat, rice, maize and Sorghum ) to assist with genetic and physical mapping and candidate gene identification in Ae . sharonensis . These resources provide an initial framework to further build on and characterise the genetic and genomic structure of Ae . sharonensis .

Natural Variation in Brachypodium Links Vernalization and Flowering Time Loci as Major Flowering Determinants

Standing genetic variation for flowering time in a nondomesticated grass encompasses known and novel regulators. The domestication of plants is underscored by the selection of agriculturally favorable developmental traits, including flowering time, which resulted in the creation of varieties with altered growth habits. Research into the pathways underlying these growth habits in cereals has highlighted the role of three main flowering regulators: VERNALIZATION1 (VRN1), VRN2, and FLOWERING LOCUS T (FT). Previous reverse genetic studies suggested that the roles of VRN1 and FT are conserved in Brachypodium distachyon yet identified considerable ambiguity surrounding the role of VRN2. To investigate the natural diversity governing flowering time pathways in a nondomesticated grass, the reference B. distachyon accession Bd21 was crossed with the vernalization-dependent accession ABR6. Resequencing of ABR6 allowed the creation of a single-nucleotide polymorphism-based genetic map at the F4 stage of the mapping population. Flowering time was evaluated in F4:5 families in five environmental conditions, and three major loci were found to govern flowering time. Interestingly, two of these loci colocalize with the B. distachyon homologs of the major flowering pathway genes VRN2 and FT, whereas no linkage was observed at VRN1. Characterization of these candidates identified sequence and expression variation between the two parental genotypes, which may explain the contrasting growth habits. However, the identification of additional quantitative trait loci suggests that greater complexity underlies flowering time in this nondomesticated system. Studying the interaction of these regulators in B. distachyon provides insights into the evolutionary context of flowering time regulation in the Poaceae as well as elucidates the way humans have utilized the natural variation present in grasses to create modern temperate cereals.

The development of quick, robust, quantitative phenotypic assays for describing the host–nonhost landscape to stripe rust

Nonhost resistance is often conceptualized as a qualitative separation from host resistance. Classification into these two states is generally facile, as they fail to fully describe the range of states that exist in the transition from host to nonhost. This poses a problem when studying pathosystems that cannot be classified as either host or nonhost due to their intermediate status relative to these two extremes. In this study, we investigate the efficacy of the Poaceae-stripe rust (Puccinia striiformis Westend.) interaction for describing the host–nonhost landscape. First, using barley (Hordeum vulgare L.) and Brachypodium distachyon (L.) P. Beauv. We observed that macroscopic symptoms of chlorosis and leaf browning were associated with hyphal colonization by P. striiformis f. sp. tritici, respectively. This prompted us to adapt a protocol for visualizing fungal structures into a phenotypic assay that estimates the percent of leaf colonized. Use of this assay in intermediate host and intermediate nonhost systems found the frequency of infection decreases with evolutionary divergence from the host species. Similarly, we observed that the pathogen’s ability to complete its life cycle decreased faster than its ability to colonize leaf tissue, with no incidence of pustules observed in the intermediate nonhost system and significantly reduced pustule formation in the intermediate host system as compared to the host system, barley-P. striiformis f. sp. hordei. By leveraging the stripe rust pathosystem in conjunction with macroscopic and microscopic phenotypic assays, we now hope to dissect the genetic architecture of intermediate host and intermediate nonhost resistance using structured populations in barley and B. distachyon.

Discovery and characterization of two new stem rust resistance genes in Aegilops sharonensis

Key messageWe identified two novel wheat stem rust resistance genes, Sr-1644-1Sh and Sr-1644-5Sh in Aegilops sharonensis that are effective against widely virulent African races of the wheat stem rust pathogen.AbstractStem rust is one of the most important diseases of wheat in the world. When single stem rust resistance (Sr) genes are deployed in wheat, they are often rapidly overcome by the pathogen. To this end, we initiated a search for novel sources of resistance in diverse wheat relatives and identified the wild goatgrass species Aegilops sharonesis (Sharon goatgrass) as a rich reservoir of resistance to wheat stem rust. The objectives of this study were to discover and map novel Sr genes in Ae. sharonensis and to explore the possibility of identifying new Sr genes by genome-wide association study (GWAS). We developed two biparental populations between resistant and susceptible accessions of Ae. sharonensis and performed QTL and linkage analysis. In an F6 recombinant inbred line and an F2 population, two genes were identified that mapped to the short arm of chromosome 1Ssh, designated as Sr-1644-1Sh, and the long arm of chromosome 5Ssh, designated as Sr-1644-5Sh. The gene Sr-1644-1Sh confers a high level of resistance to race TTKSK (a member of the Ug99 race group), while the gene Sr-1644-5Sh conditions strong resistance to TRTTF, another widely virulent race found in Yemen. Additionally, GWAS was conducted on 125 diverse Ae. sharonensis accessions for stem rust resistance. The gene Sr-1644-1Sh was detected by GWAS, while Sr-1644-5Sh was not detected, indicating that the effectiveness of GWAS might be affected by marker density, population structure, low allele frequency and other factors.

The genetic architecture of colonization resistance in Brachypodium distachyon to non-adapted stripe rust (Puccinia striiformis) isolates

Multilayered defense responses ensure that plants are hosts to only a few adapted pathogens in the environment. The host range of a plant pathogen depends on its ability to fully overcome plant defense barriers, with failure at any single step sufficient to prevent life cycle completion of the pathogen. Puccinia striiformis, the causal agent of stripe rust (=yellow rust), is an agronomically important obligate biotrophic fungal pathogen of wheat and barley. It is generally unable to complete its life cycle on the non-adapted wild grass species Brachypodium distachyon, but natural variation exists for the degree of hyphal colonization by Puccinia striiformis. Using three B. distachyon mapping populations, we identified genetic loci conferring colonization resistance to wheat-adapted and barley-adapted isolates of P. striiformis. We observed a genetic architecture composed of two major effect QTLs (Yrr1 and Yrr3) restricting the colonization of P. striiformis. Isolate specificity was observed for Yrr1, whereas Yrr3 was effective against all tested P. striiformis isolates. Plant immune receptors of the nucleotide binding, leucine-rich repeat (NB-LRR) encoding gene family are present at the Yrr3 locus, whereas genes of this family were not identified at the Yrr1 locus. While it has been proposed that resistance to adapted and non-adapted pathogens are inherently different, the observation of (1) a simple genetic architecture of colonization resistance, (2) isolate specificity of major and minor effect QTLs, and (3) NB-LRR encoding genes at the Yrr3 locus suggest that factors associated with resistance to adapted pathogens are also critical for non-adapted pathogens.