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Plant Molecular Biology

, Volume 74, Issue 3, pp 279–292 | Cite as

Functional characterization of two chimeric proteins between a Petunia inflata S-locus F-box protein, PiSLF2, and a PiSLF-like protein, PiSLFLb-S2

  • Allison M. Fields
  • Ning Wang
  • Zhihua Hua
  • Xiaoying Meng
  • Teh-hui KaoEmail author
Article

Abstract

Self-incompatible solanaceous species possess the S-RNase and SLF (S-locus F-box) genes at the highly polymorphic S-locus, and their products mediate S-haplotype-specific rejection of pollen tubes in the style. After a pollen tube grows into the style, the S-RNases produced in the style are taken up; however, only self S-RNase (product of the matching S-haplotype) can inhibit the subsequent growth of the pollen tube. Based on the finding that non-self interactions between PiSLF (Petunia inflata SLF) and S-RNase are stronger than self-interactions, and based on the biochemical properties of PiSLF, we previously proposed that a PiSLF preferentially interacts with its non-self S-RNases to mediate their ubiquitination and degradation, thereby only allowing self S-RNase to exert its cytotoxic function. We further divided PiSLF into three potential Functional Domains (FDs), FD1-FD3, based on sequence comparison of PiSLF and PiSLF-like proteins, and based on S-RNase-binding properties of these proteins and various truncated forms of PiSLF2 (S 2 allelic variant of PiSLF). In this work, we examined the in vivo function of FD2, which we proposed to be responsible for strong, general interactions between PiSLF and S-RNase. We swapped FD2 of PiSLF2 with the corresponding region of PiSLFLb-S2 (S 2 allelic variant of a PiSLF-like protein), and expressed GFP-fused chimeric proteins, named b-2-b and 2-b-2, in S 2 S 3 transgenic plants. We showed that neither chimeric protein retained the SI function of PiSLF2, suggesting that FD2 is necessary, but not sufficient, for the function of PiSLF. Moreover, since we previously found that b-2-b and 2-b-2 only interacted with S3-RNase ~50 and ~30%, respectively, as strongly as did PiSLF2 in vitro, their inability to function as PiSLF2 is also consistent with our model predicating on strong interaction between a PiSLF and its non-self S-RNases as part of the biochemical basis for S-haplotype-specific rejection of pollen tubes.

Keywords

Chimeric proteins Petunia inflata Self-incompatibility S-locus F-box protein S-RNase 

Abbreviations

FD

Functional domain

GFP

Green fluorescent protein

GST

Glutathione-S-Transferase

HMM

Hidden Markov model

NOS-ter

Transcription termination signal of the nopaline synthase gene

PP

Posterior probability

PPd

Posterior probability of divergence

PiSLF

Petunia inflataS-locus F-box protein

PiSLFL

Petunia inflataS-locus F-box like protein

pLAT52

The promoter of LAT52 of tomato

PVDF

Polyvinylidene difluoride

RT-PCR

Reverse transcription polymerase chain reaction

SCF

Skp1 Cullin-1 F-box and Rbx1

SI

Self-incompatibility

SLF

S-locus F-box protein

SLF

S-locus F-box

SR

Specific region

Notes

Acknowledgments

We thank Michael Goralczyk and Melinda Bothe for assistance in genomic DNA preparation, Jiong Wang for general laboratory help, and Anthony Omeis for greenhouse management. This work was supported by National Science Foundation grant IOS 08-43195 to T.-h.K.

Supplementary material

11103_2010_9672_MOESM1_ESM.doc (32 kb)
Supplementary material 1 (DOC 32 kb)
11103_2010_9672_MOESM2_ESM.xls (185 kb)
Supplementary material 2 (XLS 185 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Allison M. Fields
    • 1
  • Ning Wang
    • 2
  • Zhihua Hua
    • 2
    • 3
  • Xiaoying Meng
    • 2
  • Teh-hui Kao
    • 1
    • 2
    Email author
  1. 1.Department of Biochemistry and Molecular BiologyThe Pennsylvania State UniversityUniversity ParkUSA
  2. 2.Intercollege Graduate Degree Program in Plant BiologyThe Pennsylvania State UniversityUniversity ParkUSA
  3. 3.Department of GeneticsUniversity of WisconsinMadisonUSA

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