Comparison of the transcriptomes of partially resistant and highly susceptible apple cultivars in response to Neonectria ditissima infection Larisa Garkava-Gustavsson 1a , Marjan Ghasemkhani 1ɑ, Björn Canbäck 2, Jakob Willforss 1,3 , Erik Alexandersson 1,3 , Hilde Nybom4, Eric van de Weg 5, Tetyana Zhebentyayeva 6 1 Swedish University of Agricultural Sciences, Alnarp, Sweden 2 Lund University, Ecology Building, Lund, Sweden 3 PlantLink, Swedish University of Agricultural Sciences-Lund University, Sweden 4 Swedish University of Agricultural Sciences, Balsgård, Sweden 5 Wageningen University and Research , Wageningen, Netherlands 6 Clemson University, Clemson, SC, US a These authors contributed equally European canker – a devastating disease! M. Lateur • Caused by a fungus, Neonectria ditissima (formerly Nectria galligena) • Infects more than 100 species (e.g., apple, pear , birch, poppel, beach, willow, oak). Do any resistant individual of those species exist? • In apple – significant damages on trees in orchards and fruit in storage : loss of produce • Removing of canker damages is time consuming and labour intensive! • Information on the genetic control of resistance would greatly enhance the prospects for breeding resistant cultivars What mechanisms are involved in the resistance responses? Background • Resistance: highly quantitative trait, no complete resistance Approach • Reveal differences in responses between partially resistant and highly susceptible cultivars by RNAseq analysis • Here: ‘ Jonathan ’ & ‘ Prima ’ • Identify differentially expressed genes ( DEGs ) • Superimpose transcriptome data with QTL intervals previously established at WUR in ’ Jonathan ’ x ’ Prima ’ • Adequate experimental design is crucial!!! Methodology • Inoculation • N. ditissima - • Water (control) • Sampling: • Three different time points • 5 (T1) , 15 (T2) , and 30 (T3) days after inoculation • Inoculation and control • Three biological replicates / timepoint / treatment /cultivar • In total, 18 samples per cultivar • Libraries • generated with 1 µg of total RNA • using Illumina TruSeq Stranded mRNA, • multiplexed and sequenced at SciLifeLab (Stockholm, Sweden) Methodology II • High quality reads were mapped to • the reference genome of apple (v. 3.0) • the reference genome of Neonectria ditissima (New Zealand). • Filtered reads were used for further analyses • A principal component analysis (PCA) • Normalization and differential expression analysis with the DESeq2 package (FDR < 0.05) • Functional annotation based on UniProt data base • Gene ontology enrichment analysis with AgriGo (FDR < 0.05) Results • Evaluation of cultivar differences in resistances • At 5, 15 and 30 dai – no visible lesions were observed • At 101 dai: ‘ Jonathan ’ showed smaller cankers than ‘ Prima ’ (p<0.01) Confirming that ‘ Jonathan ’ has higher level of resistance Results II Fraction of reads mapped to N. ditissima increase from T1 to T3 • More pronounced expression in ’ Prima ’ • Sampling occasions chosen adequately! 400 300 200 100 Nectria Control T1 T2 T3 Cultivar responses to infection over time 20 20 10 10 0 0 Nectria -10 -10 Control PC3: 14% variance PC2: 21% variance -20 -20 -30 -20 -10 0 10 20 30 -20 -10 0 10 20 PC1: 57% variance PC2: 21% variance PCA plots illustrating relative similarity/differences among samples of infected and uninfected control tissue of ’ Jonathan ’ and ’ Prima ’ at three different time points MA plots visualizing amount of expression & changes in expression Prima T1 Jonathan T1 Prima T2 Jonathan T2 Prima T3 Jonathan T3 Responses • T1 – almost none in both cvs • T2 – ’Prima ’ < ’Jonathan ’ • T3 – ’Prima ’ >> ’ Jonathan ’ T1 T2 T3 Venn diagram showing the number of differentially expressed genes in ’Johathan’ and ’Prima’ • The number of DEG s increased in time from 4 to 7,251 to 14,020 • The contribution of the susceptible cultivar increased in time, from 46% to 64% for the 15 dai and 30 dai samples respectively Enriched genes Three Gene Ontology classes: • ‘Biological process’ In the following slides: • ‘Cellular component ’ • ‘Molecular function ’ • Statistically significantly enriched genes: related to defense strategies? Example 1: Jonathan – T2 • ’Cellular component’: only one GO term (GO:0005576) is significantly enriched • Extracellular region • Biological meaning? • Fungi-statica? • The fungus has not affected intracellular components yet? Example 2: Jonathan – T3 Multiple significantly enriched GO terms • Extracellular region • Cell structures involved in photosynthesis: chloroplast, photosystem I, plastid thylacoid, photosynthetic membrane Example 3: Prima T2 Multiple significantly enriched GO terms • Extracellular region, apoplast • External encapsulating structure, cell wall • Cell structures involved in photosynthesis: chloroplast, plastid thylacoid, stromule • Ribonucleoprotein complex, ribosome • Mitochondrial inner membrane, mitochondrial proton-transporting ATP- syntase complex Example 4: Prima T3 ’Prima’ T3 • The GO:0005576 – extracellular region is not significantly enriched any more (FDR= 0.12, ns) • Cell structures involved in photosynthesis: chloroplast, plastid thylacoid, stromule • Membrane, membrane part, intrinsic to membrane, integral to membrane Conclusions • The experimental design of this study allowed us to generate robust and biologically meaningfull transcriptomic data • We have observed cultivar differences and temporal changes in the N. ditissima – apple cultivar interactions • More effective defensive strategy seems to be the prolonged restriction of the fungus to the extracellular region Future I Compare differences in temporal changes for ’Jonathan’ and ’Prima’ involving: • Polygaracturonase-inhibiting protein genes • known to confer fungal resistance in Arabidopsis • Chitinase genes + genes of chitin signalling pathway • Phenylpropanoid biosynthesis pathway • biosynthesis SA through PAL for example • Cytochrome P450 • Jasmonic acid pathway • plant defence against necrothrophic pathogens • Ethylene pathway • plant defence, response to biotic/abiotic stress • Salicilic acid pathway • plant defence, responce biotic/abiotic stress Future II • The results of transcriptome analysis of ’Jonathan ’ and ’ Prima ’ will be superimposed with the QTL intervals • Transcriptome analyses of ’Aroma ’ and ’ Discovery ’. • ’Aroma ’ and ’ Discovery ’ are resistant and susceptible parents of a QTL mapping population • All this research aims to come to a better understanding on resistance mechanisms and resistance components Acknowledgements Co-authors: SLU SLU-LU ’PlantLink’ LU WUR Clemson Univ. Tatyana Marjan Hilde Jakob Björn Eric Erik Zhebentyayeva Ghasemkhani Nybom Alexandersson Willforss Canbäck van de Weg Financial support: .