Hypersensitive response to Potato virus Y in potato cultivar Sárpo Mira is conferred by the Ny-Smira gene located on the long arm of chromosome IX

Potato virus Y (PVY, Potyvirus) is the fifth most important plant virus worldwide in terms of economic and scientific impact. It infects members of the family Solanaceae and causes losses in potato, tomato, tobacco, pepper and petunia production. In potato and its wild relatives, two types of resistance genes against PVY have been identified. While Ry genes confer symptomless extreme resistance, Ny genes cause a hypersensitive response visible as local necrosis that may also be able to prevent the virus from spreading under certain environmental conditions. The potato cultivar Sárpo Mira originates from Hungary and is highly resistant to PVY, although the source of this resistance remains unknown. We show that cv. Sárpo Mira reacts with a hypersensitive response leading to necrosis after PVY(NTN) infection in detached leaf, whole plant and grafting assays. The hypersensitivity to PVY(NTN) segregated amongst 140 individuals of tetraploid progeny of cvs. Sárpo Mira × Maris Piper in a 1:1 ratio, indicating that it was conferred by a single, dominant gene in simplex. Moreover, we identified five DNA markers linked to this trait and located the underlying locus (Ny-Smira) to the long arm of potato chromosome IX. This position corresponds to the location of the Ry(chc) and Ny-1 genes for PVY resistance. A simple PCR marker, located 1 cM from the Ny-Smira gene, can be recommended for selection of PVY-resistant progeny of cv. Sárpo Mira

The potato NB LRR gene family. Determination, characterisation and utilisation for rapid identification of novel disease resistance genes.

*[N.B.: Additional files were attached to this thesis at the time of its submission. Please refer to the author for further details.

Identification of Avramr1 from Phytophthora infestans using long read and cDNA pathogen-enrichment sequencing (PenSeq)

Potato late blight, caused by the oomycete pathogen Phytophthora infestans, significantly hampers potato production. Recently, a new Resistance to Phytophthora infestans (Rpi) gene, Rpi‐amr1, was cloned from a wild Solanum species, Solanum americanum. Identification of the corresponding recognized effector (Avirulence or Avr) genes from P. infestans is key to elucidating their naturally occurring sequence variation, which in turn informs the potential durability of the cognate late blight resistance. To identify the P. infestans effector recognized by Rpi‐amr1, we screened available RXLR effector libraries and used long read and cDNA pathogen‐enrichment sequencing (PenSeq) on four P. infestans isolates to explore the untested effectors. Using single‐molecule real‐time sequencing (SMRT) and cDNA PenSeq, we identified 47 highly expressed effectors from P. infestans, including PITG_07569, which triggers a highly specific cell death response when transiently coexpressed with Rpi‐amr1 in Nicotiana benthamiana, suggesting that PITG_07569 is Avramr1. Here we demonstrate that long read and cDNA PenSeq enables the identification of full‐length RXLR effector families and their expression profile. This study has revealed key insights into the evolution and polymorphism of a complex RXLR effector family that is associated with the recognition by Rpi‐amr1

Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing

Global yields of potato and tomato crops are reduced owing to potato late blight disease, which is caused by Phytophthora infestans. Although most commercial potato varieties are susceptible to blight, wild potato relatives are not and are therefore a potential source of Resistance to P. infestans (Rpi) genes. Resistance breeding has exploited Rpi genes from closely related tuber-bearing potato relatives, but is laborious and slow 1–3. Here we report that the wild, diploid non-tuber-bearing Solanum americanum harbors multiple Rpi genes. We combine R gene sequence capture (RenSeq4) with single-molecule real-time SMRT sequencing (SMRT RenSeq) to clone Rpi-amr3i . This technology should enable de novo assembly of complete nucleotide-binding, leucine-rich repeat receptor (NLR) genes, their regulatory elements and complex multi-NLR loci from uncharacterized germplasm. SMRT RenSEQ can be applied to rapidly clone multiple R genes for engineering pathogen-resistant crops

Genome and time-of-day transcriptome of Wolffia australiana link morphological minimization with gene loss and less growth control.

Rootless plants in the genus Wolffia are some of the fastest growing known plants on Earth. Wolffia have a reduced body plan, primarily multiplying through a budding type of asexual reproduction. Here, we generated draft reference genomes for Wolffia australiana (Benth.) Hartog & Plas, which has the smallest genome size in the genus at 357 Mb and has a reduced set of predicted protein-coding genes at about 15,000. Comparison between multiple high-quality draft genome sequences from W. australiana clones confirmed loss of several hundred genes that are highly conserved among flowering plants, including genes involved in root developmental and light signaling pathways. Wolffia has also lost most of the conserved nucleotide-binding leucine-rich repeat (NLR) genes that are known to be involved in innate immunity, as well as those involved in terpene biosynthesis, while having a significant overrepresentation of genes in the sphingolipid pathways that may signify an alternative defense system. Diurnal expression analysis revealed that only 13% of Wolffia genes are expressed in a time-of-day (TOD) fashion, which is less than the typical ∼40% found in several model plants under the same condition. In contrast to the model plants Arabidopsis and rice, many of the pathways associated with multicellular and developmental processes are not under TOD control in W. australiana, where genes that cycle the conditions tested predominantly have carbon processing and chloroplast-related functions. The Wolffia genome and TOD expression data set thus provide insight into the interplay between a streamlined plant body plan and optimized growth

A complex resistance locus in Solanum americanum recognizes a conserved Phytophthora effector

Late blight caused by Phytophthora infestans greatly constrains potato production. Many Resistance (R) genes were cloned from wild Solanum species and/or introduced into potato cultivars by breeding. However, individual R genes have been overcome by P. infestans evolution; durable resistance remains elusive. We positionally cloned a new R gene, Rpi-amr1, from Solanum americanum, that encodes an NRC helper-dependent CC-NLR protein. Rpi-amr1 confers resistance in potato to all 19 P. infestans isolates tested. Using association genomics and long-read RenSeq, we defined eight additional Rpi-amr1 alleles from different S. americanum and related species. Despite only ~90% identity between Rpi-amr1 proteins, all confer late blight resistance but differentially recognize Avramr1 orthologues and paralogues. We propose that Rpi-amr1 gene family diversity assists detection of diverse paralogues and alleles of the recognized effector, facilitating durable resistance against P. infestans

Prediction of the tomato Ph-3 late blight disease resistance gene using public marker data and the re-annotated tomato NB-LRR complement

<p>Members of the nucleotide-binding leucine-rich repeat (NB-LRR) class of plant disease resistance (<em>R</em>) genes are key-receptors during the innate immunity response against a wide range of pests and diseases. Many efforts are therefore carried out to identify and annotate this gene family within genome sequencing projects of crop plants to, for example, provide plant breeders with a genomic toolbox.</p> <p>Genome sequencing efforts of members of the genus Solanum, which includes the crops potato and tomato, have not only aided the identification of the full <em>R</em> gene complements, but also further showed that the chromosomal location of gene subfamilies is highly conserved. In a previous publication (Andolfo et al., 2014), we exploited this fact together with publicly available genetic marker data, to predict the potential candidate for the tomato late blight <em>R</em> gene <em>Ph-3</em>. By superimposing four markers from two independent publications (Andolfo et al., 2013 and Zhang et al., 2013) over the <em>R</em> gene-annotated tomato chromosome 9, we pin-pointed to a single candidate Solyc09g092310. The recent publication of the Ph-3 sequence (Zhang et al., 2014) allowed us to compare this sequence, which is derived from <em>S. pimpinellifolium</em> L3708, with our candidate Solyc09g092310 from the tomato reference <em>S. lycopersicum</em> Heinz1706. Initial blastn searches against the Heinz1706 NB-LRR complement returned the four candidates flanked by Indel_3 and P55 (Figure A). However the highest sequence similarity with over 95% (nucleotide) over the full coding sequence was identified for our candidate Solyc09g092310 (Figure B), delimited between markers TG591 and P55.</p> <p>This result shows another important application of the reannotated NB-LRR gene complement in predicting <em>R</em> gene candidates in conjunction with previously derived genetic marker data that describe yet uncharacterised disease resistance loci.</p> <p> </p> <p> </p> <p>Andolfo G, Jupe F, Witek K, Etherington GE, Ercolano MR, Jones JDG. (2014). Defining the full tomato NB-LRR resistance gene repertoire using genomic and cDNA RenSeq. BMC Plant Biology 14(120). doi:10.1186/1471-2229-14-120</p> <p>Zhang C, Liu L, Wang X, Vossen J, Li G, Li T, Zheng Z, Gao J, Guo W, Visser RGF, Li J, Bai Y, Du Y (2014). The Ph-3 gene from Solanum pimpinellifolium encodes CC-NBS-LRR protein conferring resistance to Phytophthora infestans. Theoretical and Applied Genetics. doi:10.1007/s00122-014-2303-1</p> <p>Andolfo G, Sanseverino W, Aversano R, Frusciante L, Ercolano MR (2013) Genome-wide 
identification and analysis of candidate genes for disease resistance in 
tomato. Mol breeding, 9928:7. doi:10.1007/s11032-013-9928-7</p> <p>Zhang C, Liu L, Zheng Z, Sun Y, Zhou L, Yang Y, Cheng F, Zhang Z, Wang X, 
Huang S, Xie B, Du Y, Bai Y, Li J (2013) Fine mapping of the Ph‐3 gene conferring resistance to late blight (Phytophthora infestans) in tomato. Theor Appl Genet, 126:2643–2653. doi: 10.1007/s00122-013-2162-1</p