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Suppression of mRNAs for lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in pea reduces sensitivity to the phytotoxin coronatine and disease development by Mycosphaerella pinodes

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Abstract

Using a recently developed model pathosystem involving Medicago truncatula and Mycosphaerella pinodes, causal agent of Mycosphaerella blight on pea to understand host molecular response to a fungal suppressor, we applied the suppressor to leaves of M. truncatula and identified 151 nonredundant cDNA fragments as newly expressed genes. These included genes encoding lipoxygenase (LOX) and enoyl-CoA hydratase, which are presumably involved in jasmonic acid (JA) synthesis. Potential genes encoding plastidic enzymes, including allene oxide synthase (AOS) and allene oxide cyclase (AOC), and other peroxisomal enzymes involved in β-oxidation were predicted from the Medicago Gene Index EST database and tested for altered expression by semiquantitative RT-PCR. The coordinated expression of genes encoding both plastidic and peroxisomal enzymes showed that the suppressor likely conditions certain cellular process(es) through the JA synthesis in M. truncatula. To explore the role of JA or JA-regulated cellular process(es) in conditioning susceptibility, we used an Apple latent spherical virus (ALSV)-based virus-induced gene silencing (VIGS) technology to silence pea genes including LOX, AOS, AOC and 12-oxo-phytodienoic acid reductase (OPR). In LOX-, AOS-, AOC- or OPR-silenced pea plants, disease development induced by M. pinodes was remarkably reduced. Similarly, silencing of mRNA for LOX, AOS, AOC or OPR reduced the sensitivity to a phytotoxin, coronatine, which is believed to act through a JA-dependent process. On the basis of these results, it is conceivable that M. pinodes has evolved a strategy to condition susceptibility by manipulating the physiology of host cells, in particular JA-regulated cellular process(es), to promote disease development in pea.

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Acknowledgments

The authors are grateful to Mr. Y. Sogabe, Application Development Center, Analytical Applications Department, Shimazu Co.,Ltd., Kyoto, Japan, for technical assistance with the MultiNA analysis. We also thank Ms. M. Morizane and Mr. S. Yamamoto in our laboratory for technical assistance. This research was supported in part by the Grants-in-Aids for Scientific Research (No. 22580051) from the Japan Society for Promotion of Science (JSPS).

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Authors and Affiliations

  1. Laboratory of Plant Pathology and Genetic Engineering, Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushima-naka, Kita-ku, Okayama, 700-8530, Japan

    Kazuhiro Toyoda, Yuriko Kawanishi, Yuriko Kawamoto, Chiaki Kurihara, Yoshishige Inagaki, Yuki Ichinose & Tomonori Shiraishi

  2. Plant Pathology Laboratory, Faculty of Agriculture, Iwate University, 3-18-8 Ueda, Morioka, 020-8550, Japan

    Noriko Yamagishi, Akihiro Tamura & Nobuyuki Yoshikawa

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  1. Kazuhiro Toyoda
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  2. Yuriko Kawanishi
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  10. Tomonori Shiraishi
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Correspondence to Kazuhiro Toyoda.

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The nucleotide sequence data reported here are available in the DDBJ/EMBL/GenBank database.

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Fig. 1 Apple latent spherical virus (ALSV)-based gene silencing in pea. a Schematic diagram for inoculating ALSV vectors with pea (Pisum sativum L. cv. Puget) and its systemic infection. Stipule at first node (node 1; yellow) of 10-day-old seedlings was inoculated mechanically with a sap containing the wild-type ALSV carrying no fragment (wtALSV) and grown for 4 more weeks. The growing proximal leaflets (true leaves) at nodes 2 to 7 were tested for the systemic infection, using an RT-PCR (upper) and a tissue printing assay (lower) to detect the accumulation of ALSV-RNA-2 and its encoded coat proteins, respectively. Pea elongation factor 1-α (PsEF1-α) was used for the control amplification. b Whole view of vegetative growth (1), proximal leaflet (2), distal tendril (3), flower (3) and seed (4) development of pea seedlings taken at 4 weeks after inoculation with wild-type (wt) ALSV. Note that plants inoculated with wtALSV did not develop any viral symptoms or significant morphological changes. c Phytoenedesaturase (PDS) gene was silenced in pea seedlings infected with ALSVvector carrying a cDNA fragment for PDS gene from soybean. The soyPDS:ALSV vector containing a 300-bp fragment of soybean PDS gene homologous to positions 494–793 of soyPDS (Igarashi et al. 2009; Genbank/EMBL/DDBJ accession M64704) was used to inoculate pea seedlings in the same way, resulting in a uniform photobleached phenotype throughout plant growth. Image shows leaflets at fourth node from the wtALSV (left)- and the soyPDS:ALSV (right)-inoculated pea seedling at 3 weeks after ALSV inoculation.

Fig. 2 Silencing of lipoxygenase (LOX)-, allene oxide synthase (AOS)-, allene oxide cyclase (AOC)- and 12-oxo-phytodienoic acid reductase (OPR)-mRNA in pea plants infected with Apple latent spherical virus (ALSV) vectors. a Stipule at first node of 10-day-old pea seedlings was inoculated mechanically with sap containing the wild-type ALSV carrying no fragment (wtALSV), cDNA fragment of the LOX (ALSV:LOX), AOS (ALSV:AOS), AOC (ALSV:AOC) or OPR (ALSV:OPR) gene. Systemic infection was confirmed by RT-PCR to amplify the ALSV-RNA2, using total RNAs from fourth leaf at fourth node of growing seedlings at 2 weeks after ALSV inoculation. Elongation factor 1-α (PsEF1-α) was used for the control amplification. b Pea seedlings at 3 weeks after inoculation with the respective ALSV vectors did not have any significant morphological changes compared with mock-inoculated control plants (Mock). c Silencing of mRNA forLOX, AOS, AOC and OPR in pea plants infected with Apple latent spherical virus (ALSV) vectors. Silencing was confirmed at 2 weeks after ALSV inoculation by suppression of wound-induced accumulation for LOX-, AOS-, AOC- and OPR-mRNA using total RNAs from fourth leaves at fourth node of pea seedlings inoculated with the wild-type ALSV (NS; nonsilenced plants), ALSV containing cDNA fragment for LOX (ALSV:LOX), AOS (ALSV:AOS), AOC (ALSV:AOC) or OPR (ALSV:OPR) (S; silenced plants). Mock-inoculated plants (M) were also used as control plants.

Fig. 3 Semiquantitative RT-PCR analysis for mRNA of lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in Medicago truncatula leaves treated with the fungal suppressor, methyl jasmonate (MeJA) or phytotoxin corronatine. Leaf discs from 6-week-old seedlings of M. truncatula were treated for 1.5 h with the fungal suppressor (50, 100 and 200 μg/mL), MeJA (50, 100 and 200 μM) or coronatine (50, 100 and 200 μM). Control was treated with distilled water (0). “N” indicates amplification from untreated tissues. Primer sequences and expected size of amplified DNA are shown in supplemental Table 1. Elongation factor 1-α (EF1-α) was used for the control amplification.

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Toyoda, K., Kawanishi, Y., Kawamoto, Y. et al. Suppression of mRNAs for lipoxygenase (LOX), allene oxide synthase (AOS), allene oxide cyclase (AOC) and 12-oxo-phytodienoic acid reductase (OPR) in pea reduces sensitivity to the phytotoxin coronatine and disease development by Mycosphaerella pinodes . J Gen Plant Pathol 79, 321–334 (2013). https://doi.org/10.1007/s10327-013-0460-8

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