Scientia Agricultura Sinica ›› 2015, Vol. 48 ›› Issue (4): 683-694.doi: 10.3864/j.issn.0578-1752.2015.04.06

• PLANT PROTECTION • Previous Articles     Next Articles

Identification of a Protein both in Tobacco and Rice that Interacts with an HR-elicitor SsbX of Xanthomonas oryzae pv. oryzicola

SUN Yu-jing, MA Wen-xiu, CAI Lu-lu, LIU Liang, ZOU Li-fang, CHEN Gong-you   

  1. School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai 200240
  • Received:2014-08-26 Online:2015-02-16 Published:2015-02-16

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Abstract: 【Objective】A highly-conserved single-stranded DNA-binding protein (SsbX) of Xanthomonas oryzae pv. oryzicola, secreted through the type-III secretion system (T3SS), elicits hypersensitive response (HR) in non-host tobacco. The objective of this study is to clarify how it triggers HR. 【Method】 The appropriate period of rice and tobacco leaves were infiltrated with X. oryzae pv. oryzicola RS105 strain to construct rice and tobacco cDNA libraries using CreatorTM SMARTTM cDNA Library Construction Kit, then the qualities of rice and tobacco cDNA libraries were determined by using the special enzyme cutting site SfiⅠof pGADT- SfiAB vector. These cDNA libraries were applied to screen SsbX-interacting proteins on SD/-Ade/-Leu/-Trp/-His medium by yeast two-hybrid system (Y2H). The homology and phylogenetic tree analysis of SsbX-interacting proteins were operated by the method of neighbor-joining using MEGA.4 software. The sublocalization of SsbX with SsbX-interacting proteins in tobacco leaves was observed and imaged under a laser confocal microscope at 488 nm, the excitation wavelength for YFP, and 520 to 550 nm, the emission filter wavelength.【Result】Construction and detection of rice and tobacco cDNA libraries showed the average length of cDNAs inserted into pGADT-SfiAB was 1.0 kb among 20 randomly-selected rice cDNA library clones and 1.2 kb among 20 tobacco cDNA library clones, indicating good qualities of rice and tobacco cDNA libraries could be applied for subsequent research. The Y2H and β-gal assay results demonstrated that SsbX interacted with an actin-depolymerization factor 2 (ADF2) either from rice or tobacco and this couple made AH109 positively grow on SD/-Ade/-Leu/-Trp/-His plate with blue color. Hereby this factor was designated OsADF2 for rice and NbADF2 for tobacco correspondingly. OsADF2 encoded a 139 amino acid (aa) protein, 15.9 kD in molecular weight, and NbADF2 encoded a 137 aa in 15.8 kD. These two proteins were highly conserved with 69.02% identities to each other in protein sequences. Homology and phylogenetic tree analysis displayed that ADF2 proteins in plants were highly conserved in similarity up to 60%. NbADF2 had 69.05% similarities to the homolog of Arabidopsis, higher than orthologs in other dicotyledonous plants. OsADF2 showed 88.81% similarities to the homolog of Setaria italic, higher than orthologs in other monocots plant. Bimolecular fluorescence complementation (BiFC) assay results demonstrated that the presence of OsADF2 or NbADF2 together with SsbX, like a positive control, displayed yellow fluorescence in plant plasma membrane under a laser confocal microscope when the pair proteins were transciently expressed in plant cells mediated by Agrobacterium. 【Conclusion】 The SsbX protein of Xanthomonas, secreted through the T3SS, targets ADF2 in plant plasma membrane to trigger plant immunity. This novel finding provides a scientific clue to further understand how SsbX elicits HR in tobacco plants.

Key words: Xanthomonas oryzae pv. oryzicola, single-stranded DNA-binding protein (SsbX), actin-depolymerization factor 2 (ADF2), yeast two-hybrid, bimolecular fluorescence complementation, hypersensitive response

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