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Journal of General Plant Pathology

, Volume 83, Issue 3, pp 140–146 | Cite as

Improved method for in situ biolistic transformation to analyze barley–powdery mildew interactions

  • Miki Wahara
  • Chie Inoue
  • Tomohiro Kohguchi
  • Koreyuki Sugai
  • Kappei Kobayashi
  • Masamichi Nishiguchi
  • Naoto Yamaoka
  • Takashi YaenoEmail author
Fungal Diseases
  • 323 Downloads

Abstract

Although the use of stable transformants is indispensable to elucidate mechanisms underlying molecular plant–pathogen interactions, this approach remains difficult to apply to crops. Alternatively, biolistic transformation has often been used as a transient expression method in various plants. In this study, we developed a method for in situ biolistic transformation without separating leaves from barley seedlings by using a hand-held particle bombardment system because unwounded leaves are preferable for analyzing interactions between barley and Blumeria graminis f. sp. hordei which requires healthy living cells. As a result, we found that the infection rate in intact leaves was higher than in separated leaves and that the transformation efficiencies in leaves were higher when plants were grown in vermiculite rather than in culture soil. Furthermore, we determined the appropriate inoculation time after bombardment to analyze the incompatible interaction and successfully monitored the gradual occurrence of cell death over time. Our system was suitable for relatively long-term follow-up analysis of the fate of each single cell during plant–pathogen interactions.

Keyword

Blumeria graminis Biolistic bombardment HR cell death 

Notes

Acknowledgements

We thank Dr. Beat Keller (University of Zürich) and Dr. Tsuyoshi Nakagawa (Shimane University) for kindly providing the plasmids pV26-UMUG and pGWBs, respectively. This work was supported by the Inamori Foundation, Institute for Fermentation Osaka and JSPS KAKENHI Grant Number JP24780046 and JP16K07618.

Supplementary material

10327_2017_712_MOESM1_ESM.pdf (431 kb)
Supplementary material 1 (PDF 430 KB)

References

  1. Bieri S, Mauch S, Shen QH, Peart J, Devoto A, Casais C, Ceron F, Schulze S, Steinbiß HH, Shirasu K, Schulze-Lefert P (2004) RAR1 positively controls steady state levels of barley MLA resistance proteins and enables sufficient MLA6 accumulation for effective resistance. Plant Cell 16:3480–3495CrossRefPubMedPubMedCentralGoogle Scholar
  2. Douchkov D, Nowara D, Zierold U, Schweizer P (2005) A high-throughput gene-silencing system for the functional assessment of defense-related genes in barley epidermal cells. Mol Plant-Microbe Interact 18:755–761CrossRefPubMedGoogle Scholar
  3. Hu P, Meng Y, Wise RP (2009) Functional contribution of chorismate synthase, anthranilate synthase, and chorismate mutase to penetration resistance in barley–powdery mildew interactions. Mol Plant-Microbe Interact 22:311–320CrossRefPubMedGoogle Scholar
  4. Jones JDG, Dangl JL (2006) The plant immune system. Nature 444:323–329CrossRefPubMedGoogle Scholar
  5. Nowara D, Gay A, Lacomme C, Shaw J, Ridout C, Douchkov D, Hensel G, Kumlehn J, Schweizer P (2010) HIGS: host-induced gene silencing in the obligate biotrophic fungal pathogen Blumeria graminis. Plant Cell 22:3130–3141CrossRefPubMedPubMedCentralGoogle Scholar
  6. Pliego C, Nowara D, Bonciani G, Gheorghe DM, Xu R, Surana P, Whigham E, Nettleton D, Bogdanove AJ, Wise RP, Schweizer P, Bindschedler LV, Spanu PD (2013) Host-induced gene silencing in barley powdery mildew reveals a class of ribonuclease-like effectors. Mol Plant-Microbe Interact 26:633–642CrossRefPubMedGoogle Scholar
  7. Seeholzer S, Tsuchimatsu T, Jordan T, Bieri S, Pajonk S, Yang W, Jahoor A, Shimizu KK, Keller B, Schulze-Lefert P (2010) Diversity at the Mla powdery mildew resistance locus from cultivated barley reveals sites of positive selection. Mol Plant-Microbe Interact 23:497–509CrossRefPubMedGoogle Scholar
  8. Shirasu K, Nielsen K, Piffanelli P, Oliver R, Schulze-Lefert P (1999) Cell-autonomous complementation of mlo resistance using a biolistic transient expression system. Plant J 17:293–299CrossRefGoogle Scholar
  9. Sugai K, Masaoka H, Penjore K, Hanboonsong Y, Nishiguchi M, Yamaoka N (2010) The time and spatial strategy of Blumeria graminis f. sp. hordei for surviving after failure of first infection. Physiol Mol Plant Pathol 74:346–350CrossRefGoogle Scholar
  10. Wang MB, Abbott DC, Upadhyaya NM, Jacobsen JV, Waterhouse PM (2001) Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Aust J Plant Physiol 28:149–156Google Scholar
  11. Ward SV, Manners JG (1974) Environmental effects on the quantity and viability of conidia produced by Erysiphe graminis. Trans Br Mycol Soc 62: 119–128CrossRefGoogle Scholar
  12. Wei F, Wing RA, Wise RP (2002) Genome dynamics and evolution of the Mla (powdery mildew) resistance locus in barley. Plant Cell 14:1903–1917CrossRefPubMedPubMedCentralGoogle Scholar
  13. Yaeno T, Matsuda O, Iba K (2004) Role of chloroplast trienoic fatty acids in plant disease defense responses. Plant J 40:931–941CrossRefPubMedGoogle Scholar
  14. Yaeno T, Saito B, Katsuki T, Iba K (2006) Ozone-induced expression of the Arabidopsis FAD7 gene requires salicylic acid, but not NPR1 and SID2. Plant Cell Physiol 47:355–362CrossRefPubMedGoogle Scholar
  15. Zhou F, Kurth J, Wei F, Elliott C, Valè G, Yahiaoui N, Keller B, Somerville S, Wise R, Schulze-Lefert P (2001) Cell-autonomous expression of barley Mla1 confers race-specific resistance to the powdery mildew fungus via a Rar1-independent signaling pathway. Plant Cell 13:337–350CrossRefPubMedPubMedCentralGoogle Scholar
  16. Zipfel C, Robatzek S (2010) Pathogen-associated molecular pattern-triggered immunity: veni, vidi…? Plant Physiol 154:551–554CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© The Phytopathological Society of Japan and Springer Japan 2017

Authors and Affiliations

  1. 1.Department of AgricultureEhime UniversityMatsuyamaJapan
  2. 2.Research Unit for Plasma Medicine, Agriculture and FisheryEhime UniversityMatsuyamaJapan
  3. 3.Research Unit for CitromicsEhime UniversityMatsuyamaJapan

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