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.

Development and validation of allele specificRLKgene markers for banana bunchy top disease (BBTD) resistance in banana

“…These preliminary findings may help us better understand the impact of these mutations on the gene's ability to combat banana bunchy top disease. However, it is vital to underline the importance of additional functional studies such as gene silencing, knockout, and overexpression (Bao et al 2022) to further support and validate this inference.…”

Genome-wide Identification of the Aconitase Gene Family in Tomato (<em>Solanum lycopersicum</em>) and CRISPR-based Functional Characterization of <em>SlACO2</em> on Male-sterility

“…In a recent study using AMS, another gene encoding a basic helix-loop-helix (bHLH) TF was knocked out by CRISPR/Cas9 and over-expressed in tomato. SlAMS downregulation or upregulation led to abnormal pollen development, which in turn decreased pollen viability, and subsequently generated male-sterile lines [5]. In our laboratory, we have generated completely male-sterile lines by mutating the SlPHD_MS1 gene (Solyc04g008420), encoding a PHD-type transcription factor [20].…”

“…In these studies, pollen development at tetrad, post-meiotic and mature stages were investigated in different tissues, including microspores, generative cells, pollen cells, callus, fully expanded leaves, pistil, and mature pollen grain (Figure S6). SlACO genes, four genes related to male sterility; SlPHD_MS1 [20], SlMS10 [21], SlAMS [5], SlSTR1 [22], and these 25 genes were hierarchically clustered by Manhattan distance in three anatomical parts including microspores, generative cell and pollen cell (Figure S6). Three sub-clusters were observed, and the SlACO2 gene was found to cluster separately from SlACO1.…”

“…The failure of plants to produce functional anthers, pollen, or male gametes, which is also known as male sterility (MS), can be induced by genic or cytoplasmic systems, chemical hybridizing agents, or transgenic technology in plants. Incorporating male sterility into tomato breeding is a critical task to reduce the cost of hybrid seed production and ensure high varietal purity [5].…”

The tapetal tissue is essential for the maintenance of redox homeostasis during microgametogenesis in tomato

“…The Solyc01g081100 gene is a homolog of the Arabidopsis bHLH10/89/90 gene that, together with DYT1‐MYB35, forms a regulatory module during tapetum and pollen development (Cui et al., 2016 ). Recently, an ABORTED MICROSPORES ( AMS ) tomato homolog was identified that is essential for pollen development in this species (Bao et al., 2022 ).…”