Genotype-specific suppression of multiple defense pathways in apple root during infection by Pythium ultimum

Abstract

The genotype-specific defense activation in the roots of perennial tree crops to soilborne necrotrophic pathogens remains largely unknown. A recent phenotyping study indicated that the apple rootstock genotypes B.9 and G.935 have contrasting resistance responses to infection by Pythium ultimum. In the current study, a comparative transcriptome analysis by Illumina Solexa HiSeq 3000 platform was carried out to identify the global transcriptional regulation networks between the susceptible B.9 and the resistant G.935 to P. ultimum infection. Thirty-six libraries were sequenced to cover three timepoints after pathogen inoculation, with three biological replicates for each sample. The transcriptomes in the roots of the susceptible genotype B.9 were reflected by overrepresented differentially expressed genes (DEGs) with downregulated patterns and systematic suppression of cellular processes at 48 h post inoculation (hpi). In contrast, DEGs with annotated functions, such as kinase receptors, MAPK signaling, JA biosynthesis enzymes, transcription factors, and transporters, were readily induced at 24 hpi and continued up-regulation at 48 hpi in G.935 roots. The earlier and stronger defense activation is likely associated with an effective inhibition of necrosis progression in G.935 roots. Lack of effector-triggered immunity or existence of a susceptibility gene could contribute to the severely disturbed transcriptome and susceptibility in B.9 roots. The identified DEGs constitute a valuable resource for hypothesis-driven studies to elucidate the resistance/tolerance mechanisms in apple roots and validating their potential association with resistance traits.

References

[1] Jaffee, B., Abawi, G. & Mai, W. Role of soil microflora and Pratylenchus penetrans in an apple replant disease. Phytopathology 72, 247-251 (1982).

[2] Jaffee, B., Abawi, G. & Mai, W. Fungi associated with roots of apple seedlings grown in soil from an apple replant site. Plant Dis. 66, 942-944 (1982).

[3] Mazzola, M. Elucidation of the microbial complex having a causal role in the development of apple replant disease in Washington. Phytopathology 88, 930-938 (1998).

[4] Mazzola, M. Identification and pathogenicity of Rhizoctonia spp. isolated from apple roots and orchard soils. Phytopathology 87, 582-587 (1997).

[5] Tewoldemedhin, Y. T., Mazzola, M., Botha, W. J., Spies, C. F. & McLeod, A. Characterization of fungi (Fusarium and Rhizoctonia) and oomycetes (Phytophthora and Pythium) associated with apple orchards in South Africa. Eur. J. Plant Pathol. 130, 215-229 (2011).

[6] Covey, R. P., Benson, N. R. & Haglund, W. A. Effect of soil fumigation on the apple replant disease in Washington. Phytopathology 69, 684-686 (1979).

[7] Fazio, G., Aldwinckle, H., Robinson, T. & Wan, Y. Implementation of molecular marker technologies in the apple rootstock breeding program in Geneva-challenges and successes. Acta Horticulturae 9031, 61-68 (2011).

[8] Boller, T. & Felix, G. A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals by pattern-recognition receptors. Annu. Rev. Plant Biol. 60, 379-406 (2009).

[9] Dodds, P. N. & Rathjen, J. P. Plant immunity: towards an integrated view of plant-pathogen interactions. Nat. Rev. Genet. 11, 539-548 (2010).

[10] Dangl, J. L., Horvath, D. M. & Staskawicz, B. J. Pivoting the plant immune system from dissection to deployment. Science 341, 746-751 (2013).

[11] Bonardi, V. & Dangl, J. L. How complex are intracellular immune receptor signaling complexes?. Front. Plant Sci. 3, 237 (2012).

[12] Chisholm, S. T., Coaker, G., Day, B. & Staskawicz, B. J. Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124, 803-814 (2006).

[13] Jones, J. D. & Dangl, J. L. The plant immune system. Nature 444, 323 (2006).

[14] Boller, T. & He, S. Y. Innate immunity in plants: an arms race between pattern recognition receptors in plants and effectors in microbial pathogens. Science 324, 742-744 (2009).

[15] Glazebrook, J. Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu. Rev. Phytopathol. 43, 205-227 (2005).

[16] Luna, E. et al. Callose deposition: a multifaceted plant defense response. Mol. Plant-Microbe Interact. 24, 183-193 (2011).

[17] Mengiste, T. Plant immunity to necrotrophs. Annu. Rev. Phytopathol. 50, 267-294 (2012).

[18] Tsuda, K., Sato, M., Glazebrook, J., Cohen, J. D. & Katagiri, F. Interplay between MAMP‐triggered and SA‐mediated defense responses. Plant J. 53, 763-775 (2008).

[19] Moore, J. W., Loake, G. J. & Spoel, S. H. Transcription dynamics in plant immunity. Plant Cell 23, 2809-2820 (2011).

[20] Ramirez, S. R. & Basu, C. Comparative analyses of plant transcription factor databases. Curr. Genom. 10, 10-17 (2009).

[21] Riechmann, J. L. et al. Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290, 2105-2110 (2000).

[22] De Geyter, N., Gholami, A., Goormachtig, S. & Goossens, A. Transcriptional machineries in jasmonate-elicited plant secondary metabolism. Trends Plant Sci. 17, 349-359 (2012).

[23] Grayer, R. J. & Kokubun, T. Plant-fungal interactions: the search for phytoalexins and other antifungal compounds from higher plants. Phytochemistry 56, 253-263 (2001).

[24] Rushton, D. L. et al. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol. J. 10, 2-11 (2012).

[25] Mikulic-Petkovsek, M., Stampar, F. & Veberic, R. Seasonal changes in phenolic compounds in the leaves of scab-resistant and susceptible apple cultivars. Can. J. Plant Sci. 89, 745-753 (2009).

[26] Zhu, Y., Shao, J., Zhou, Z. & Davis, R. E. Comparative transcriptome analysis reveals a preformed defense system in apple root of a resistant genotype of G. 935 in the absence of pathogen. Int. J. Plant Genom. 2017, 8950746 (2017).

[27] Shin, S. et al. Transcriptome changes specifically associated with apple (Malus domestica) root defense response during Pythium ultimum infection. Physiol. Mol. Plant Pathol. 94, 16-26 (2016).

[28] Zhu, Y., Shin, S. & Mazzola, M. Genotype responses of two apple rootstocks to infection by Pythium ultimum causing apple replant disease. Canadian J. Plant Pathol. 38, 483-491 (2016).

[29] Weiss, S., Bartsch, M. & Winkelmann, T. Transcriptomic analysis of molecular responses in Malus domestica ‘M26’ roots affected by apple replant disease. Plant Mol. Biol. 94, 303-318 (2017).

[30] Martin, L., Fei, Z., Giovannoni, J. & Rose, J. K. C. Catalyzing plant science research with RNA-seq. Front. Plant Sci. 4, 66 (2013).

[31] Metzker, M. L. Sequencing technologies-the next generation. Nat. Rev. Genet. 11, 31 (2010).

[32] Wang, Z., Gerstein, M. & Snyder, M. RNA-Seq: a revolutionary tool for transcriptomics. Nat. Rev. Genet. 10, 57 (2009).

[33] Delteil, A. et al. Several wall-associated kinases participate positively and negatively in basal defense against rice blast fungus. BMC Plant Biol. 16, 17 (2016).

[34] Li, H., Zhou, S.-Y., Zhao, W.-S., Su, S.-C. & Peng, Y.-L. A novel wall-associated receptor-like protein kinase gene, OsWAK1, plays important roles in rice blast disease resistance. Plant Mol. Biol. 69, 337-346 (2009).

[35] Bouwmeester, K. & Govers, F. Arabidopsis L-type lectin receptor kinases: phylogeny, classification, and expression profiles. J. Exp. Bot. 60, 4383-4396 (2009).

[36] Singh, P. & Zimmerli, L. Z. Lectin receptor kinases in plant innate immunity. Front. Plant Sci. 4, 124 (2013).

[37] Colcombet, J. & Hirt, H. Arabidopsis MAPKs: a complex signalling network involved in multiple biological processes. Biochem. J. 413, 217-226 (2008).

[38] Liu, Y. & Zhang, S. Phosphorylation of 1-aminocyclopropane-1-carboxylic acid synthase by MPK6, a stress-responsive mitogen-activated protein kinase, induces ethylene biosynthesis in Arabidopsis. Plant Cell 16, 3386-3399 (2004).

[39] Pitzschke, A., Schikora, A. & Hirt, H. MAPK cascade signalling networks in plant defence. Curr. Opin. Plant Biol. 12, 421-426 (2009).

[40] Zhang, Y., Goritschnig, S., Dong, X. & Li, X. A gain-of-function mutation in a plant disease resistance gene leads to constitutive activation of downstream signal transduction pathways in suppressor of npr1-1, constitutive 1. Plant Cell 15, 2636-2646 (2003).

[41] van Schie, C. C. & Takken, F. L. Susceptibility genes 101: how to be a good host. Annu. Rev. Phytopathol. 52, 551-581 (2014).

[42] Robert-Seilaniantz, A., Navarro, L., Bari, R. & Jones, J. D. Pathological hormone imbalances. Curr. Opin. Plant Biol. 10, 372-379 (2007).

[43] Browse, J. Jasmonate passes muster: a receptor and targets for the defense hormone. Annu. Rev. Plant Biol. 60, 183-205 (2009).

[44] Yang, S. F. & Hoffman, N. E. Ethylene biosynthesis and its regulation in higher plants. Annu. Rev. Plant Physiol. 35, 155-189 (1984).

[45] Asai, T. et al. MAP kinase signalling cascade in Arabidopsis innate immunity. Nature 415, 977 (2002).

[46] Jonak, C., Ökrész, L., Bögre, L. & Hirt, H. Complexity, cross talk and integration of plant MAP kinase signalling. Curr. Opin. Plant Biol. 5, 415-424 (2002).

[47] Nakagami, H., Pitzschke, A. & Hirt, H. Emerging MAP kinase pathways in plant stress signalling. Trends Plant Sci. 10, 339-346 (2005).

[48] Nühse, T. S., Peck, S. C., Hirt, H. & Boller, T. Microbial elicitors induce activation and dual phosphorylation of the Arabidopsis thaliana MAPK 6. J. Biol. Chem. 275, 7521-7526 (2000).

[49] Mao, G. et al. Phosphorylation of a WRKY transcription factor by two pathogen-responsive MAPKs drives phytoalexin biosynthesis in Arabidopsis. Plant Cell 23, 1639-1653 (2011).

[50] Ren, D. et al. A fungal-responsive MAPK cascade regulates phytoalexin biosynthesis in Arabidopsis. Proc. Natl Acad. Sci. 105, 5638-5643 (2008).

[51] Thomma, B. P., Nürnberger, T. & Joosten, M. H. Of PAMPs and effectors: the blurred PTI-ETI dichotomy. Plant Cell 23, 4-15 (2011).

[52] Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357 (2012).

[53] Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).

[54] Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403-410 (1990).

[55] Hulsen, T., de Vlieg, J. & Alkema, W. BioVenn-a web application for the comparison and visualization of biological lists using area-proportional Venn diagrams. BMC Genom. 9, 488 (2008).

[56] Zhou, Z., Cong, P., Tian, Y. & Zhu, Y. Using RNA-seq data to select reference genes for normalizing gene expression in apple roots. PLoS ONE 12, e0185288 (2017).

[57] Livak, K. J. & Schmittgen, T. D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔCT method . Methods 25, 402-408 (2001).