Facilitating smart crop breeding through understanding the genetics of resistance and virulence in the Striga-rice interaction

Lead Research Organisation: University of Sheffield
Department Name: Animal and Plant Sciences

Abstract

Summary (4000 characters)
Rice is one of the most important crops worldwide and plays a pivotal role in the national economies of sub-Saharan Africa (SSA) yet yields are constrained by species of obligate root parasitic weeds called Striga that cause yield losses of 20-100%. Striga is extremely difficult to control as individuals produce millions of small, long-lived, genetically diverse seeds that only germinate in response to germination stimulants present in host root exudates. As Striga severely damages the development of the crop immediately after attaching to the host root, control measures must act early in the life cycle to prevent losses in yield. Resistant varieties should play a key role in control strategies yet there is no published information about the identity or mechanism of action of Striga-resistance genes. Striga evolves rapidly to overcome host resistance, strongly influencing the sustainability of crop improvement strategies based on breeding for resistance. Stacking many resistance genes in a variety is essential to reduce the likelihood of resistance being overcome by the evolution of the parasite but this requires knowledge about the identity of host resistance and parasite virulence genes. This project aims to discover new resistance genes and to link this with an understanding the molecular genetic basis of parasite virulence genes for breeding of durable defence. We have recently identified the first cluster of resistance genes (Receptor-Like-Proteins (RLP)) on chromosome 12 of rice that provide resistance against several ecotypes (genetic variants) of S. hermonthica and S. asiatica and shown that simultaneous down-regulation of many of these genes in a resistant cultivar (by RNAi) restores susceptibility. However, for effective breeding of varieties, we need to know whether one, or more of the genes, acts to provide resistance, or whether different combinations of genes act against different genetic ecotypes of the parasite. We will investigate this by inactivating RLP genes, singly and in combination using CRISPR/Cas9 in resistant varieties, infecting with different ecotypes of Striga, and determining the effect on susceptibility. Recently we have compared Striga growing on susceptible and resistant hosts and taken a population genomics approach to identify a set of candidate virulence genes. To determine whether they interact with the RLP resistance genes we will collect Striga ecotypes from multiple host species/varieties across SSA. These will be genotyped with 'neutral' markers (SNPs) to look at the structure of the populations, (critical for interpretation and management of virulence) and then with SNPs in candidate virulence genes to test for specific associations with known forms of resistance. This will provide a foundation for future studies on the functional interaction between resistance and virulence genes and for association analyses to detect virulence genes relevance to other sets of resistance genes. To discover new resistance genes we will use a rice (Nested Association Mapping (NAM)) population derived from crossing one common O. sativa ssp indica parent (cv. IR64) with ten diverse parents. Two thousand NAM lines have been genotyped to produce highly detailed genetic maps for each of the 10 populations, facilitating the rapid discovery of new Striga resistance genes. We will first phenotype the 10 parents with existing and new Striga ecotypes to form a matrix of virulence/resistance to allow us to select interesting ecotypes and NAM populations to phenotype during the 2-year project whilst providing data for more extensive phenotyping in the field and laboratory in the future.

Technical Summary

Striga hermonthica (Sh) and S. asiatica (Sa) are obligate root parasitic weeds that parasitize rainfed rice in Africa causing devastating yield losses. To enable effective breeding of durable defence to multiple ecotypes of Striga, we need to stack resistance genes with different modes of action in rice varieties. To prolong the longevity of resistance, knowledge of the evolutionary potential and population genetics of parasite virulence is crucial. We have identified the first cluster of Receptor-Like-Protein resistance genes that provide resistance against several ecotypes of Sh and Sa and shown that by down-regulating many of these genes simultaneously in the resistant variety Nipponbare, that full susceptibility is restored. To identify exactly which gene or genes are responsible we will utilize CRISPR/Cas9 gene editing to knock out specific genes, singly and in combination and test their effect on susceptibility to different ecotypes of Striga. We will characterise the variation of recently identified candidate Sh virulence loci (from a single Sh ecotype from Kibos, Kenya) in other Sh ecotypes from different regions of Africa. For each ecotype, we will infect the same resistant and susceptible varieties used originally, collect 50 individual Sh plants that attach and grow and genotype them for 100 SNPs, including 50 virulence candidates and 50 control SNPs. This will reveal the population structure of Sh ecotypes, essential for understanding the evolution of virulence alleles and determine how our candidate Sh virulence loci are distributed in different ecotypes. We will also identify, for the first time, candidate virulence loci from Sa ecotypes using both population genomic and quantitative genetic approaches. Finally, we will phenotype a novel, Nested Association Mapping population of rice for resistance to Sa and Sh ecotypes to discover multiple new resistance genes with different modes of action for breeding elite varieties with durable resistance.

Planned Impact

Our goal is to undertake research that will facilitate the production of rice varieties that are resistant to Striga and produce high yields. We will take a multi-disciplinary approach involving molecular, quantitative and population genetics that both builds on and advances previous work to identify resistance genes in rice and candidate virulence loci in Striga to elucidate the molecular genetic basis of host-parasite specificity. This knowledge will facilitate the future development of a rice-Striga interaction model for predictive breeding of resistance in elite varieties and a rice variety 'decision tool' to advise farmers which varieties to grow in different areas. The expected impacts of this project consist of academic knowledge, applied tools, techniques and methodologies of immediate use to plant breeders and capacity building activities to disseminate information and establish new contacts in Africa. This project will provide new insights into biology of the plant-plant interaction and will advance our understanding of the interaction between virulence genes and resistance alleles. The discovery of new resistance genes and QTL, together with high-resolution marker data, will enhance the effectiveness of the genetics and the breeding pipeline through the use of marker-assisted selection. Such information will be made available to plant breeders via our established relationship with Africa Rice. The project will provide the basis for CRISPR-(Cas9) strategies to quickly develop resistant varieties from elite lines already highly performing but susceptible to Striga, a potential breakthrough for breeding. Identification of new resistance genes will allow us to explore the O. sativa genetic diversity of the genes through allelic analysis of the publically available 3,000 genomes data potentially allowing the discovery of new or rare alleles for breeding resistance. The long-term impact is to facilitate 'smart' breeding and effective use of varieties with appropriate combinations of resistance genes to protect crops against different genetic variants of the parasite, ultimately improving rice yields and food security for resource for poor farmers. This will be achieved through follow-on projects and field-testing of elite varieties. Prof Scholes and Dr Rodenburg already have established field sites for testing varieties for resistance to Striga in Uganda, Tanzania and Uganda. By partnering with African-based organizations, like the Africa Rice Center (AfricaRice), the knowledge and products derived from the project will be readably available to African rice breeding programs. They have already shown a keen interest for implementation. AfricaRice has an impressive track record in breeding and delivering improved rice varieties in the region. The path from gene-discovery to variety adoption and increased food security and/or income generation is rather long but the Africa-based partner in this project, AfricaRice, has extensive experience with this and a clear strategy. Africa Rice is coordinating an effective African-wide Rice Breeding Task Force (RBTF) of more than twenty African countries, involving national agricultural research systems (NARS), most of which effectively reach out to national extension and seed systems.

Publications

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Description Striga hermonthica and S. asiatica are obligate parasitic weeds that parasitize the roots of cereals in sub Saharan Africa (SSA) causing enormous yield losses worth > 7 billion US$ annually. Rice is one of the world's most important crops and the most rapidly expanding in SSA but production is constrained by Striga infestations in 38 African countries, leading to region- wide annual production losses of 0.5 M tons of grain worth 199 million US$. Effective control of Striga is essential for food security but it remains elusive. The use of Striga-resistant varieties should form the cornerstone of control programs, as resistance is recognized as sustainable and cost effective for farmers. We have shown that when resistant rice cultivars are grown in the field in Africa, that yields can be greatly improved but the use of resistant cultivars is constrained by our limited of knowledge of the identity, genetic basis and mode of action of genes underlying resistance to Striga and by the potential for rapid evolution of virulence in the parasite.

The current GCRF project aims to (a) determine the function of a known cluster of Striga resistance genes in rice that provides broad-spectrum resistance against a range of genetic ecotypes of S. hermonthica (b) discover new S. asiatica-resistance genes by phenotyping mapping populations of rice developed by Dr Mathias Lorieux (CIAT, Colombia) and (c) begin the identification of virulence loci in S. hermonthica and S. asiatica using a range of genomic approaches.

We recently identified the first cluster of resistance genes (Receptor-Like- Proteins (RLP)) in rice that provide resistance against several ecotypes of S. hermonthica but we don't know whether one, or more of the genes, acts to provide resistance, or whether different combinations of genes act against different genetic ecotypes of the parasite. Thus the first aim of the GCRF grant is to knock out each RLP gene in the resistant cultivar IR64 using CRISPR/Cas9 technology to see if the cultivar becomes susceptible to S. hermonthica and to express each resistance gene in a susceptible cultivar to see if the cultivar becomes resistant to S. hermonthica. This work is progressing very well. The PDRA, Dr Emily Beardon, has made all the over-expression constructs at the University of Sheffield and she is currently working in the rice transformation unit at CIAT Colombia (with our collaborator) to produce the CRISPR constructs (half are complete) and to transform the resistant and susceptible cultivars with the overexpression and CRISPR constructs. To date all the over-expression constructs have been transformed into Azucena.

To discover new resistance genes to S. asiatica, we have phenotyped the 10 parents of a NAM mapping population for resistance to 4 different S. asiatica ecotypes (two from Ethiopia, one from Tanzania and one from USA). We now have a matrix of virulence/resistance that has allowed us to select an appropriate mapping population to phenotype for resistance to one of the ecotype of S. asiatica from Ethiopia. The phenotyping of resistance in the mapping population is underway and initial analysis of the resistance/susceptibility of the first set of lines is very promising as, Striga-resistance QTL are emerging. This work is in progress and will take a further 6 months to complete.

In order to begin the identification of virulence loci in different S. asiatica ecotypes, the four ecotypes that showed different virulence profiles on the 10 rice cultivars of the NAM population have been selected. We have extracted DNA from an individual of each ecotype and sequenced their genomes. The genome sequences have been aligned and are now being subjected to different comparative genomic analyses to determine how different the sequences are from each other and whether we can detect differences in candidate virulence loci. In addition, we have collected 13 different ecotypes of S. asiatica from different regions of Madagascar and we are currently infecting rice cultivars to allow us to collect leaf material from individuals within ecotypes for sequencing. Seeds from the individuals will also be collected to allow us to characterize their virulence profiles on different rice hosts. When all the individuals have been sequenced (which will take ~ 12 months) we will analyse the data to produce SNP markers that will ultimately allow is to carry out a Genome Wide Association Analysis to identify virulence loci. Recently we compared S. hermonthica individuals (from a population of seeds collected from Kibos in Kenya) growing on a susceptible rice host with the few individuals that can infect a strongly resistance host and taken a population genomics approach to identify the 'virulence' genes which allow some Striga individuals to overcome the resistance in the normally resistant cultivar. This work was carried out as part of a previous SCPRID project and we have now completed the population genetic analyses as part of the GCRF grant. The results are exciting and have allowed us to identify a set of candidate virulence genes underlying differences in pathogenicity. These genes are being investigated further and we are in process of writing a manuscript.
Exploitation Route Striga is a problem unique to Africa and leads to devastating yield losses in rain fed rice affecting the lives of millions of resource poor small-holder farmers. Good control of Striga has not yet been achieved and reducing yield losses due to Striga is essential for food security and poverty alleviation. The use of resistant cultivars in Striga control programs is limited by lack of knowledge about the identity and genetics of both host resistance genes and parasite virulence genes that constantly evolve to overcome host resistance. Breeders need to be able to stack genes within farmer preferred varieties knowing that they will protect the crop against the genetic ecotype(s) of Striga in that region. This proposal will, for the first time, link the identification and function of novel Striga resistance genes in rice with the identification an understanding of the genetics of parasite virulence genes to facilitate predictive breeding of cultivars with durable resistance for use in different agro-ecological zones. The ability to breed varieties with durable resistance to Striga is a low cost, sustainable strategy for farmers and would have a great impact on both stabilising and increasing yields thus promoting the economic welfare of small holder farmers across sub Saharan Africa, key aims of ODA funding in LMICs.

The current project will provide new insights into biology of the Striga-plant interaction and identify and begin to validate candidate Striga resistance genes. In addition, we will identify Striga virulence alleles and gain information about their interaction with Striga resistance genes. This information will ultimately facilitate the development of an 'interaction model' between virulence genes and resistance alleles (though more extensive work and identification of both resistance genes and virulence alleles in populations of Striga work will be required). The aim is to produce a map of geographic distribution of virulence alleles, associated with the resistance genes as this will eventually permit the development of a software tool for farmers that will suggest the best varieties to cultivate according to location.
Sectors Agriculture, Food and Drink,Education

 
Description The project has only been running for one year. Thus, the non-academic impacts of the current GCRF grant are limited to capacity building and education. This GCRF grant builds of previous work on Striga resistance funded by the BBSRC SCPRID programme. In November 2017, Professor Scholes attended a workshop at the ILRI-BECA Hub in Nairobi on Renewed Strategies for Striga Management in Africa, attended by a range of different stakeholders, young scientists and students. She presented work from the current GCRF grant on why knowledge about both host resistance to Striga and Striga virulence genes, is very important in the context of durable control of this parasitic weed. As part of the visit Prof Scholes has small group discussions with students and plant breeders about different aspects Striga biology as it relates to control strategies.
First Year Of Impact 2017
Sector Education
 
Description Government of Thailand PhD Scholarship
Amount
Organisation Government of Thailand 
Sector Public
Country Thailand
Start 02/2017 
End 02/2020
 
Description Collaboration with AfricaRice Centre 
Organisation Africa Rice Centre
Country Nigeria 
Sector Charity/Non Profit 
PI Contribution Prof Scholes works with AfricaRice scientists to field test rice cultivars and germplasm (identified as showing resistance to Striga under laboratory conditions) in the field in Africa. In previous projects field trials have been carried out in farmers fields in Tanzania and Uganda and in Kenya at the ICIPE research centre at Mbita Point. In the current GCRF project 'Facilitating smart crop breeding through understanding the genetics of resistance and virulence in the Striga-rice interaction' Prof Scholes is working with Dr Jonne Rodenburg and his team to collect seeds of different genetic ecotypes of S. asiatica and S. hermonthica for use in laboratory studies to determine the virulence profiles of the different ecotypes across different rice germplasm. Such information is essential for predictive breeding of durable defence against Striga ecotypes in different agro-ecological regions of Africa.
Collaborator Contribution Working with scientists at the AfricaRice Centre allows Prof Scholes to carry out fieldwork with farmers in Africa to identify germplasm resistance to different ecotypes of the parasite for immediate use by farmers and also to test germplasm identified as resistant in the laboratory. AfricaRice scientists also facilitate capacity building activities including the interaction and dissemination of information to young African scientists/PhD students.
Impact Seven different ecotypes of S. asiatica have been collected from Madagascar to study their virulence profiles on different rice cultivars and for use in studies to identify Striga virulence genes.
Start Year 2017
 
Description Collaboration with the International Center for Tropical Agriculture (CIAT) 
Organisation CGIAR
Department International Center for Tropical Agriculture
Country Colombia 
Sector Charity/Non Profit 
PI Contribution Prof Julie Scholes has worked with Dr Mathias Lorieux (Head of the Rice Genetics Laboratory at CIAT, Colombia) on the discovery of QTL and genes underlying resistance to the parasitic weed Striga since 2010. Professor Scholes research group exploits physiological, genomic and comparative genomic technologies to understand the mechanisms underlying susceptibility/resistance in cereals to the parasitic weed Striga and brings this expertise to the collaboration with Dr Lorieux and his team. Several years ago Prof Scholes developed a soil-free rhizotron root chamber that allows accurate phenotyping of post attachment resistance in cereals to Striga thus allowing the use of mapping populations for the identification of QTL and thus genes underlying resistance to this parasite. Prof Scholes group has used this system to to phenotype a number of mapping populations produced by Dr Lorieux (where the parents derive from African germplasm or germplasm suitable for use in Africa) to successfully identify major QTL underlying Striga resistance. This collaboration has been and continues to be very successful. Transformation Centre at CIAT, Columbia during a 6 month visit by the PDRA (see below).
Collaborator Contribution Dr Mathias Lorieux, holds a joint position at CIAT, Colombia and IRD (Institut de Recherche pour le Développement, Montpellier, France). He is based at CIAT where he leads the Rice Genetics and Genomics group. His research activities are focussed on developing methods and tools to facilitate and improve the use of wild germplasm in plant breeding. In 2008, Dr Lorieux initiated the construction of a rice Nested Association Mapping Population (NAM) of rice. The populations consist of 20 inter-subspecific crosses involving one common O. sativa spp. indica parent (IR64) and 20 O. sativa spp. japonica parents from Asia, Africa and Latin America. All populations have high density molecular marker maps which facilitates rapid QTL and gene discovery. Prof Scholes group have utilised several of the RIL populations to map resistance to Striga and are currently using this resource as part of the GCRF grant 'Facilitating smart crop breeding through understanding the genetics of resistance and virulence in the Striga-rice interaction'. In addition to the provision of rice germplasm Dr Lorieux contributes expertise in genetics analysis of the data and he has developed software (MapDisto) for analysis of different mapping populations. CIAT also has an outstanding rice transformation facility and as part of our current collaboration the PDRA working on the GCRF grant is spending time at CIAT transforming rice cultivars to knock out candidate resistance genes. Prof Scholes and Dr Lorieux are co-supervising a PhD student and postdoc as part of the GCRF grant.
Impact We have successfully mapped a major QTL for Striga hermonthica resistance in a RIL population (from the NAM population) and we are currently producing constructs to over-express and knock out (by CRISPR) candidate Striga resistance genes to determine which gene or combination of genes underlies the resistance. We are currently phenotying another RIL mapping population to identify QTL underlying resistance to S. asiatica.
Start Year 2017