Comparison of the transcriptomes of partially resistant and highly susceptible cultivars in response to 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 , (formerly galligena) • Infects more than 100 species (e.g., apple, pear , , 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 ().

• 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: