Allele mining in solanum: conserved homologues of Rpi-blb 1 are identified in Solanum stoloniferum

Allele mining facilitates the discovery of novel resistance (R) genes that can be used in breeding programs and sheds light on the evolution of R genes. Here we focus on two R genes, Rpi-blb1 and Rpi-blb2, originally derived from Solanum bulbocastanum. The Rpi-blb1 gene is part of a cluster of four paralogues and is flanked by RGA1-blb and RGA3-blb. Highly conserved RGA1-blb homologues were discovered in all the tested tuber-bearing (TB) and non-tuber-bearing (NTB) Solanum species, suggesting RGA1-blb was present before the divergence of TB and NTB Solanum species. The frequency of the RGA3-blb gene was much lower. Interestingly, highly conserved Rpi-blb1 homologues were discovered not only in S. bulbocastanum but also in Solanum stoloniferum that is part of the series Longipedicellata. Resistance assays and genetic analyses in several F1 populations derived from the relevant late blight resistant parental genotypes harbouring the conserved Rpi-blb1 homologues, indicated the presence of four dominant R genes, designated as Rpi-sto1, Rpi-plt1, Rpi-pta1 and Rpi-pta2. Furthermore, Rpi-sto1 and Rpi-plt1 resided at the same position on chromosome VIII as Rpi-blb1 in S. bulbocastanum. Segregation data also indicated that an additional unknown late blight resistance gene was present in three populations. In contrast to Rpi-blb1, no homologues of Rpi-blb2 were detected in any material examined. Hypotheses are proposed to explain the presence of conserved Rpi-blb1 homologues in S. stoloniferum. The discovery of conserved homologues of Rpi-blb1 in EBN 2 tetraploid species offers the possibility to more easily transfer the late blight resistance genes to potato varieties by classical breeding

Debate on the Exploitation of Natural Plant Diversity to Create Late Blight Resistance in Potato

This paper reports on a debate on intriguing propositions relating to the scientific, agronomic, societal and economic impact of the BIOEXPLOIT project, focusing on late blight resistance in potato. It discusses (i) whether identifying pathogen effectors will facilitate selecting durable R genes, (ii) whether breeding for durable late blight resistance requires deploying Rpi (for Resistance to P hytophthora i nfestans) genes, (iii) whether breeding strategies and cultural practices determine the durability of new resistance genes, (iv) whether marker-assisted breeding for Phytophthora infestans resistance is already in the stage of adoption, (v) to what extent genetically-modified organism technology can advance realizing late-blight resistant potato cultivars, and (vi) whether modifying R genes will result in novel broad spectrum resistanc

High resolution mapping of a novel late blight resistance gene Rpi-avll, from the wild Bolivian species Solanum avilesii

Both Mexico and South America are rich in Solanum species that might be valuable sources of resistance (R) genes to late blight (Phytophthora infestans). Here, we focus on an R gene present in the diploid Bolivian species S. avilesii. The genotype carrying the R gene was resistant to eight out of 10 Phytophthora isolates of various provenances. The identification of a resistant phenotype and the generation of a segregating population allowed the mapping of a single dominant R gene, Rpi-avl1, which is located in an R gene cluster on chromosome 11. This R gene cluster is considered as an R gene “hot spot”, containing R genes to at least five different pathogens. High resolution mapping of the Rpi-avl1 gene revealed a marker co-segregating in 3890 F1 individuals, which may be used for marker assisted selection in breeding programs and for further cloning of Rpi-avl

Societal Costs of Late Blight in Potato and Prospects of Durable Resistance Through Cisgenic Modification

In the European Union almost 6 Mha of potatoes are grown representing a value of close to ¿6,000,000,000. Late blight caused by Phytophthora infestans causes annual losses (costs of control and damage) estimated at more than ¿1,000,000,000. Chemical control is under pressure as late blight becomes increasingly aggressive and there is societal resistance against the use of environmentally unfriendly chemicals. Breeding programmes have not been able to markedly increase the level of resistance of current potato varieties. New scientific approaches may yield genetically modified marker-free potato varieties (either trans- and/or cisgenic, the latter signifying the use of indigenous resistance genes) as improved variants of currently used varieties showing far greater levels of resistance. There are strong scientific investments needed to develop such improved varieties but these varieties will have great economic and environmental impact. Here we present an approach, based on (cisgenic) resistance genes that will enhance the impact. It consists of five themes: the detection of R-genes in the wild potato gene pool and their function related to the various aspects in the infection route and reproduction of the late blight causing pathogen; cloning of natural R-genes and transforming cassettes of single or multiple (cisgenic) R-genes into existing varieties with proven adaptation to improve their value for consumers; selection of true to the wild type and resistant genotypes with similar qualities as the original variety; spatial and temporal resistance management research of late blight of the cisgenic genetically modified (GM) varieties that contain different cassettes of R-genes to avoid breaking of resistance and reduce build-up of epidemics; communication and interaction with all relevant stakeholders in society and transparency in what research is doing. One of the main challenges is to explain the different nature and possible biological improvement and legislative repercussions of cisgenic GM-crops in comparison with transgenic GM-crops. It is important to realize that the present EU Directive 2001/18/EC on GM crops does not make a difference between trans- and cisgenes. These rules were developed when only transgenic GM plants were around. We present a case arguing for an updating and refinement of these rules in order to place cisgenic GM-crops in another class of GM-plants as has been done in the past with (induced) mutation breeding and the use of protoplast fusion between crossable species. Keywords Cisgenesis - Cloning - Communication - Late blight - Phytophthora infestans - Potato - Resistance management - Selection - Transformatio

Monitoring the Dutch Phytophtora infestans population for virulence against new R-genes

New possibilities offered by marker assisted breeding and GMO breeding have sparked renewed international efforts to breed for durable potato late blight resistance. Phytophthora infestans is however known for its adaptability, a trait confirmed by recent discoveries on the structure of the P. infestans genome. One of the possibilities to enhance the durability of newly introduced host resistance is to monitor the pathogen population for virulence to new R genes, prior to - and after their introduction. The late blight control strategy should be adapted accordingly. The Dutch P. infestans population was monitored during the growing seasons 2006 – 2008. P. infestans isolates were collected from blighted production fields and from bait fields in which R gene containing potato clones were grown without fungicide protection. A selection of the P. infestans isolates collected were characterized for virulence to a range of new R genes using a detached leaf bio-assay. Virulence for all single R genes tested was found. When we focus on R genes Rpi-blb1 and Rpi-blb2, no virulence was found in 2006. One Rpi-blb1 virulent isolate was found in 2007. Another 2007 isolate was found to be virulent to Rpi-blb2. Depending on the genetic background in which Rpi-blb1 was placed 13 or 21 isolates were virulent in 2008. Depending on the genetic background in which Rpi-blb2 was placed 4 or 11 isolates were virulent in 2008. One isolate was found to infect the stacked Rpi-blb1 and Rpi-blb2 resistance genes in a detached leaf assay. From these findings it is recommended that monitoring systems should be part of future potato late blight control strategies. The resulting information on the dynamics of virulence within the local P. infestans population can then be used to enhance the durability of newly introduced host resistanc

miRNA/phasiRNA mediated regulation of plant defense response against P. syringae

Gene silencing is a mechanism of regulation of gene expression where the small RNAs (sRNAs) are key components for giving specificity to the system. In plants, two main types of noncoding small RNA molecules have been found: microRNAs (miRNAs) and small interfering RNAs (siRNAs). DCL proteins acting on large RNA precursors produce the mature forms of sRNAs (20-24nt) that can act as negative regulators of gene expression. In recent years, the role of miRNAs in regulation of gene expression in plant responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between expression profiles of different Arabidopsis thaliana mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, as differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes. We have validated this regulation, and have also established that the corresponding pri-miRNA is down-regulated upon PAMPs or bacteria perception. Using GUS reporters, we have characterized the expression pattern of both pri-miRNA and its best target R genes. We demonstrate that plants with altered levels of miRNA* (knockdown or overexpression lines) exhibit altered PTI-associated phenotypes, supporting a role for this miRNA* in the defence response against this bacterial pathogen. Finally, we identify phasiRNAs that arise from the transcript of one of the R target genes in a miRNA*-RDR6-DCL4-dependent manner.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech

PAMP-triggered immunity against Pseudomonas syringae involves microRNA-mediated regulation of several uncharacterized R genes

Two main types of noncoding small RNA molecules have been found in plants: microRNAs (miRNAs) and small interfering RNAs (siRNAs). They differ in their biogenesis and mode of action, but share similar sizes (20-24 nt). Their precursors are processed by Dicer-Like RNase III (dcl) proteins present in Arabidopsis thaliana, and in their mature form can act as negative regulators of gene expression, being involved in a vast array of plant processes, including plant development, genomic integrity or response to stress. Small-RNA mediated regulation can occurs at transcriptional level (TGS) or at post-transcriptional level (PTGS). In recent years, the role of gene silencing in the regulation of expression of genes related to plant defence responses against bacterial pathogens is becoming clearer. Comparisons carried out in our lab between the expression profiles of different mutants affected in gene silencing, and plants challenged with Pseudomonas syringae pathovar tomato DC3000, led us to identify a set of uncharacterized R genes, belonging to the TIR-NBS-LRR gene family, differentially expressed in these conditions. Through the use of bioinformatics tools, we found a miRNA* of 22 nt putatively responsible for down-regulating expression of these R genes through the generation of siRNAs. We have also found that the corresponding pri-miRNA is down-regulated after PAMP-perception in a SA-dependent manner. We also demonstrate that plants with altered levels of miRNA* (knockdown lines or overexpression lines) exhibit altered PTI-associated phenotypes, suggesting a role for this miRNA* in this defence response against bacteria. In addition we identify one of the target genes as a negative regulator of defence response against Pseudomonas syringae.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. MINECO, FEDE

Interfamily Transfer of Dual NB-LRR Genes Confers Resistance to Multiple Pathogens

A major class of disease resistance (R) genes which encode nucleotide binding and leucine rich repeat (NB-LRR) proteins have been used in traditional breeding programs for crop protection. However, it has been difficult to functionally transfer NB-LRR-type R genes in taxonomically distinct families. Here we demonstrate that a pair of Arabidopsis (Brassicaceae) NB-LRR-type R genes, RPS4 and RRS1, properly function in two other Brassicaceae, Brassica rapa and Brassica napus, but also in two Solanaceae, Nicotiana benthamiana and tomato (Solanum lycopersicum). The solanaceous plants transformed with RPS4/RRS1 confer bacterial effector-specific immunity responses. Furthermore, RPS4 and RRS1, which confer resistance to a fungal pathogen Colletotrichum higginsianum in Brassicaceae, also protect against Colletotrichum orbiculare in cucumber (Cucurbitaceae). Importantly, RPS4/RRS1 transgenic plants show no autoimmune phenotypes, indicating that the NB-LRR proteins are tightly regulated. The successful transfer of two R genes at the family level implies that the downstream components of R genes are highly conserved. The functional interfamily transfer of R genes can be a powerful strategy for providing resistance to a broad range of pathogens

Molecular characterization of cDNA encoding resistance gene-like sequences in Buchloe dactyloides

Current knowledge of resistance (R) genes and their use for genetic improvement in buffalograss (Buchloe dactyloides [Nutt.] Engelm.) lag behind most crop plants. This study was conducted to clone and characterize cDNA encoding R gene-like (RGL) sequences in buffalograss. This report is the first to clone and-characterize of buffalograss RGLs. Degenerate primers designed from the conserved motifs of known R genes were used to amplify RGLs and fragments of expected size were isolated and cloned. Sequence analysis of cDNA clones and analysis of putative translation products revealed that most encoded amino acid sequences shared the similar conserved motifs found in the cloned plant disease resistance genes RPS2, MLA6, L6, RPM1, and Xa1. These results indicated diversity of the R gene candidate sequences in buffalograss. Analysis of 5' rapid amplification of cDNA ends (RACE), applied to investigate upstream of RGLs, indicated that regulatory sequences such as TATA box were conserved among the RGLs identified. The cloned RGL in this study will further enhance our knowledge on organization, function, and evolution of R gene family in buffalograss. With the sequences of the primers and sizes of the markers provided, these RGL markers are readily available for use in a genomics-assisted selection in buffalograss