CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata
CRISPR/Cas9-mediated knockout of PiSSK1 reveals essential role of S-locus F-box protein-containing SCF complexes in recognition of non-self S-RNases during cross-compatible pollination in self-incompatible Petunia inflata
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DOI：
10.1007/s00497-017-0314-1
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发表时间：
2018-06
影响因子：
3.4
通讯作者：
Linhan Sun;T. Kao
中科院分区：
文献类型：
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作者：
Linhan Sun;T. Kao
矮牵牛自交不亲和性（self-incompatibility，SI）是矮牵牛自交不亲和性的一种抑制机制，它由多个花粉特异的S-locus F-box（SLF）基因和一个雌蕊特异的S-RNase基因组成，S2和S3单倍型具有相同的17个SLF基因（命名为SLF 1至SLF 17），并且花粉中产生的每个SLF蛋白被组装成SCF（Skp 1-Cullin 1-F-box）E3泛素连接酶复合物。一套完整的SLF蛋白被认为是集体与所有非自我S-RNases相互作用，介导其泛素化和降解的26 S蛋白酶体，允许交叉兼容授粉。对于每个SCFSLF复合物，Cullin 1亚基（命名为PiCUL 1-P）和Skp 1亚基（命名为PiSSK 1），就像F-box蛋白亚基（SLF）一样，是花粉特异性的，这提高了它们也进化为在SI中特异性功能的可能性。在这里，我们使用CRISPR/Cas9介导的基因组编辑来在PiSSK 1中产生移码插入缺失突变，并检查了花粉基因组中具有双等位基因突变的T0植物（S2 S3）和两个后代植物（S2 S2）的SI行为，每个后代植物对于一个插入缺失等位基因是纯合的，并且不携带含Cas9的T-DNA。它们的花粉与其它7种S-基因型的雌蕊完全不亲和，但与一个S_3S_3转基因植株的雌蕊完全亲和，其中S_3-RNase的产生被反义S_3-RNase基因完全抑制，与未成熟花蕾的雌蕊完全亲和，而S_3-RNase的产生很少。这些结果表明，PiSSK 1具体功能SI和支持的假设，SLF含有SCF复合物是必不可少的兼容授粉。
Key messageFunction ofPetuniaPiSSK1.AbstractSelf-incompatibility (SI), an inbreeding-preventing mechanism, is regulated inPetunia inflataby the polymorphicS-locus, which houses multiple pollen-specificS-locus F-box(SLF) genes and a single pistil-specificS-RNasegene.S2-haplotype andS3-haplotype possess the same 17 polymorphicSLFgenes (namedSLF1toSLF17), and each SLF protein produced in pollen is assembled into an SCF (Skp1–Cullin1–F-box) E3 ubiquitin ligase complex. A complete suite of SLF proteins is thought to collectively interact with all non-self S-RNases to mediate their ubiquitination and degradation by the 26S proteasome, allowing cross-compatible pollination. For each SCFSLFcomplex, the Cullin1 subunit (named PiCUL1-P) and Skp1 subunit (named PiSSK1), like the F-box protein subunits (SLFs), are pollen-specific, raising the possibility that they also evolved specifically to function in SI. Here we used CRISPR/Cas9-meditated genome editing to generate frame-shift indel mutations inPiSSK1and examined the SI behavior of aT0plant (S2S3) with biallelic mutations in the pollen genome and two progeny plants (S2S2) each homozygous for one of the indel alleles and not carrying theCas9-containing T-DNA. Their pollen was completely incompatible with pistils of seven otherwise-compatibleS-genotypes, but fully compatible with pistils of anS3S3transgenic plant in which production of S3-RNase was completely suppressed by an antisenseS3-RNasegene, and with pistils of immature flower buds, which produce little S-RNase. These results suggest that PiSSK1 specifically functions in SI and support the hypothesis that SLF-containing SCF complexes are essential for compatible pollination.