Gene silencing, knockout and over-expression of a transcription factor ABORTED MICROSPORES (SlAMS) strongly affects pollen viability in tomato (Solanum lycopersicum)

Abstract

Background: The tomato (Solanum lycopersicum L.) is an economically valuable crop grown worldwide. Because the use of sterile males reduces the cost of F1 seed production, the innovation of male sterility is of great significance for tomato breeding. The ABORTED MICROSPORES gene (AMS), which encodes for a basic helix-loop-helix (bHLH) transcription factor, has been previously indicated as an essential gene for tapetum development in Arabidopsis and rice. To determine the function of the SlAMS gene (AMS gene from S. lycopersicum) and verify whether it is a potential candidate gene for generating the male sterility in tomato, we used virus-induced gene silencing (VIGS), CRISPR/Cas9-mediated genome editing and over-expression technology to transform tomato via Agrobacterium infection.

Results: Here, the full-length SlAMS gene with 1806 bp from S. lycopersicum (Accession No. MK591950.1) was cloned from pollen cDNA. The results of pollen grains staining showed that, the non-viable pollen proportions of SlAMS-silenced (75%), -knockouted (89%) and -overexpressed plants (60%) were significantly higher than the wild type plants (less than 10%; P < 0.01). In three cases, the morphology of non-viable pollen grains appeared tetragonal, circular, atrophic, shriveled, or otherwise abnormally shaped, while those of wild type appeared oval and plump. Furthermore, the qRT-PCR analysis indicated that SlAMS in anthers of SlAMS-silenced and -knockouted plants had remarkably lower expression than in that of wild type (P < 0.01), and yet it had higher expression in SlAMS-overexpressed plants (P < 0.01).

Conclusion: In this paper, Our research suggested alternative approaches to generating male sterility in tomato, among which CRISPR/Cas9-mediated editing of SlAMS implied the best performance. We also demonstrated that the downregulation and upregulation of SlAMS both affected the pollen formation and notably led to reduction of pollen viability, suggesting SlAMS might be essential for regulating pollen development in tomato. These findings may facilitate studies on clarifying the SlAMS-associated molecular regulatory mechanism of pollen development in tomato.

Keywords: Gene knockout; Over-expression; SlAMS; Tomato; VIGS.

Fig. 1

Electrophoresis gels image showing (a) RT-PCR cloning of the tomato Aborted Microspores (SlAMS) gene and (b) recombinant colony detection. Lane M: DL-2000 DNA marker; Lane 1: negative control; Lane 2: PCR products of SlAMS gene; Lanes 3–7: colonies

Fig. 2

Phylogenetic tree among Aborted Microspores (AMS) proteins from various plant species. Plant families are shown in different colors: Solanaceae (red); Compositae (yellow); Cruciferae (blue); Cucurbitaceae (green); and Leguminosae (orange). Scale bar = 0.10 substitution rate

Fig. 3

Characterization of the tomato Aborted Microspores (SlAMS) protein. a Amino acid composition. b Predicted subcellular location. c Secondary structure. d Tertiary structure

Fig. 4

Pollen viability and morphology of the SlAMS-silenced tomato plants. a. The performance of flowers of SlAMS-silenced, −knockouted and -overexpressed plants. b. Pollen grain staining after VIGS-mediated gene silencing of SlAMS. pTRV2-SlAMS transformed; pTRV2 basic vector transformed (negative); Untransformed (WT). Blue-stained pollen grains are non-viable; colorless pollen grains are viable. c. The non-viable pollen grains percentage of SlAMS-silenced, −knockouted and -overexpressed plants and their corresponding wild type plants. **represents significance difference at 0.01 level (P < 0.01). Data are shown as means±SD (n = 3). d. Scanning electron microscope (SEM) images of pollen grains of SlAMS-silenced, −knockouted and overexpressed plants. Untransformed wild type (WT, oval). SlAMS silencing (showing tetragonal and sunken shapes). SlAMS knockout (showing round or shriveled shapes, from CR-T0–5). SlAMS overexpression (showing rhombic and other deformities, from OV-T0–3)

Fig. 5

Pollen viability and morphology of tomato plants in different SlAMS modification modes. a. PCR Identification of positive plants transformed with the pCRISPR/Cas9-SlAMS vector. Lanes 1–38: PCR products of Npt II; Lane B: no target DNA; Lane N: nontransgenic plant; Lane P: pCRISPR/Cas9-SlAMS plasmid. b. Pollen viability test after SlAMS knockout (CR-T0–2) and overexpression (OV-T0–10). Blue-stained pollen grains are non-viable; colorless pollen grains are viable. c. Scanning electron microscope (SEM) images of tomato pollen grains. Wild type (WT, oval shape); pCRISPR/Cas9-SlAMS (showing shrinkage and diamond-like shapes, from CR-T0–2). pCAMBIA2301-SlAMS (showing shriveled and atrophic shapes, from OV-T0–10); d. Target site mutation examination in CRISPR/Cas9-mediated SlAMS plant. Target shows the editing site sequence. PAM indicates the adjacent motif of the protospacer sequence. CR-ams1 indicated the first type of mutation; CR-ams2 indicated the second type; CR-ams3 indicated the third type

Fig. 6

Relative expression analysis of SlAMS-silenced, −knockouted and -overexpressed tomato plant by qRT-PCR at tetrad and maturity stage. WT represents wild type anther. VIGS represents SlAMS silencing anther, GKO represents SlAMS knockouting anther, GOE represents SlAMS overexpression anther. Suffix ‘I’ and ‘II’ indicate tetrad stage and maturity stage respectively. Data are presented as means±SD (n = 3), **indicates significant differences at P < 0.01