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.
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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
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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.
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PiSLF1 (AY500390); PiSLF2 (AY500391); PiSLF3 (AY500392); PiSLFLa-S1 (EF614190); PiSLFLa-S2 (EF614189); PiSLFLb-S2 (EF614188); PiSLFLc-S1 (EF614191); PiSLFLd-S2 (EF614187); S1(S2)A113 (AY363970AY363971); S3A113 (AY363972); S1A134 (AY363973); S2A134 (AY363974); S3A134(AY363975).
Allison M. Fields and Ning Wang contributed equally to this work.
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Fields, A.M., Wang, N., Hua, Z. et al. Functional characterization of two chimeric proteins between a Petunia inflata S-locus F-box protein, PiSLF2, and a PiSLF-like protein, PiSLFLb-S2 . Plant Mol Biol 74, 279–292 (2010). https://doi.org/10.1007/s11103-010-9672-x
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DOI: https://doi.org/10.1007/s11103-010-9672-x