CRISPR/Cas9 targeted mutagenesis of SlLBD40, a lateral organ boundaries domain transcription factor, enhances drought tolerance in tomato

Highlights

• SlLBD40 belongs to subfamily II of the LBD family of genes in tomato and was highly expressed in roots and fruit.

• SlLBD40 showed an early response to PEG and NaCl treatment.

• SlLBD40 expression was dependent on JA signaling and it might be downstream of SlMYC2.

• Compared with overexpressing transgenic and WT tomato plants, knockout of SlLBD40 by CRISPR/Cas9 enhanced the drought tolerance of tomato.

Abstract

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD)-containing genes are plant-specific genes that play important roles in lateral organ development. In this study, we identified LBD40 (Solyc02g085910), which belongs to subfamily II of the LBD family of genes in tomato. LBD40 was highly expressed in roots and fruit. LBD40 expression was significantly induced by PEG and salt. Moreover, SlLBD40 expression was induced by methyl jasmonate treatment, while SlLBD40 expression could not be induced in the jasmonic acid-insensitive1 (jai1) mutant or MYC2-silenced plants, in which jasmonic acid (JA) signaling was disrupted. These findings demonstrate that SlLBD40 expression was dependent on JA signaling and that it might be downstream of SlMYC2, which is the master transcription factor in the JA signal transduction pathway. Overexpressing and CRISPR/Cas9 mediated knockout transgenic tomato plants were generated to explore SlLBD40 function. The drought tolerance test showed that two SlLBD40 knockout lines wilted slightly, while SlLBD40 overexpressing plants suffered severe wilting. The statistical water loss rate and midday leaf water potential also confirmed that knockout of SlLBD40 improved the water-holding ability of tomato under drought conditions. Taken together, our study demonstrates that SlLBD40, involved in JA signaling, was a negative regulator of drought tolerance and that knockout of SlLBD40 enhanced drought tolerance in tomato. This study also provides a novel function of SlLBD40, which belongs to subfamily II of LBD genes.

Introduction

Water scarcity is one of the most destructive abiotic stressors in agriculture, as it seriously reduces crop productivity [1]. Drought greatly affects plant growth, weakens photosynthesis, accelerates the accumulation of reactive oxygen species, disturbs cellular homeostasis, and even causes death [[1], [2], [3]]. Roots are the primary organ responsible for drought stress, as they soak up water from soil. Photosynthetic production decreases in plants suffering from drought and is preferentially allocated to the roots, which improves the root/shoot ratio and helps roots absorb water from the soil [4]. Lateral root growth is inhibited to promote development of the primary root [5].

As a widely grown vegetable crop, tomato has gained popularity for its good nutrition and taste. However, the yield and quality of tomato are severely influenced by a myriad of abiotic stressors. As tomato is sensitive to water supply, drought is the primary growth limiting factor, particularly at the seed germination and seedling stages.

The LATERAL ORGAN BOUNDARIES DOMAIN (LBD) protein family encodes a conserved and plant-specific lateral organ boundaries (LOB) domain [6,7]. There are 42 LBD genes in the Arabidopsis genome and 46 in tomato, which have been assigned to two subfamilies [8,9]. In tomato, subfamily I is comprised of 40 genes consisting of a four-Cys motifs (C-motif), a Gly-Ala-Ser block (GAS-block), and a Leu zipper-like motif (L-motif), while subfamily II includes six genes only containing the C-motif [7,8]. It has been reported that the C-motif is required for the capacity to bind to the promoter region of downstream genes. The GAS-block appears to assist the C-motif in binding to the promoter region, and the L-motif is involved in protein-protein interactions [6,10,11].

LBD genes were initially found to be expressed in cells of the lateral organs, including the shoot apical meristem and lateral roots of Arabidopsis [7]. Subsequent studies reported that a number of LBD factors participate in the formation of lateral roots. For instance, the transcription factor AtLBD16, an auxin-inducible protein, targets PUCHI for lateral root initiation in Arabidopsis [12]. The LBD gene OsARL1, which is an auxin responsive gene, is required to initiate the formation of adventitious root primordia in rice [13]. All pollen is aborted in the lbd10 and lbd27 Arabidopsis double mutants, indicating that AtLBD10 and AtLBD27 may play a critical role in pollen development [14]. Moreover, recent studies show that the LBD gene family also participates in the stress response. Expression of VvLBD01, VvLBD02, VvLBD04, VvLBD08, and VvLBD18 in grape is involved in the responses to NaCl, mannitol, heat stress, and low temperature treatments [15]. In soybean, ninety LBD homologous genes were identified, among which the GmLBD12 was induced by various stresses, including drought stress [16]. Coincidentally, proteomic analysis showed that, in rice, LBD proteins were downregulated in Semi-rolled leaf1, 2 (SRL1 and SRL2) mutant with increased drought tolerance during drought stress, indicating that LBD proteins may be involved in drought response in rice [17]. AtLBD20 is a negative regulator that responds to Fusarium Wilt in Arabidopsis. Knockout of AtLBD20 enhances tolerance to Fusarium infection, while overexpression of AtLBD20 makes plants susceptible [18]. Nevertheless, the direct relationship between LBD factors and drought stress is unknown, and the effect of the LBD transcription factor family on abiotic resistance in tomato remains unknown as well.

The jasmonic acid-insensitive1 (jai1) is a JA-insensitive mutant in tomato, which has lost the function of the tomato orthologue of CORONATINE-INSENSITIVE1 due to a 6.2-kb deletion and fails to express JA-responsive genes [19]. Therefore, many studies have been conducted using the mutant to explore the mechanisms involved in plant growth, development, and defense related to the JA signaling pathway [[20], [21], [22], [23]]. In this study, the mutant was used to determine whether SlLBD40 is involved in JA signaling in tomato. MYC2, a basic helix-loop-helix transcription factor, mediates various JA responses in the JA signaling pathway, including inhibition of root growth, apical hook formation in the dark, leaf senescence, and defense against herbivores and pathogenic fungi [24]. In this study, we detected SlLBD40 expression in SlMYC2-silenced plants, which were obtained by virus-induced gene silencing (VIGS), to explore whether SlLBD40 expression was affected by SlMYC2.

VIGS is an RNA-mediated post-transcriptional gene silencing method. It functions as an antivirus defense mechanism to downregulate gene expression in plants [25]. It is a widely used reverse and forward-genetics method to explore gene function because of its ability to rapidly degrade mRNA of the target gene, and is simple to manipulate. VIGS has been successfully used in many species, including eggplant [26], pepper [27], strawberry [28], sweet cherry [29], cotton [30], barley [31], and potato [32]. In our previous studies, VIGS was used to verify the function of SlHSP40 in tomato [33]. VIGS has been employed to verify the heat tolerance function of SoHSC70, since it is difficult to study gene function using the transgenic method in spinach [34]. VIGS makes it possible to study gene function in situ. Furthermore, a VIGS cDNA library has been applied during fast-forward genetic screens for genes of various physiological responses. This high-throughput approach provides a large-scale phenotypic analysis and simple, fast identification of the gene responsible for the phenotype of interest [35,36]. In this study, we silenced SlMYC2 in tomato plants by VIGS and detected SlLBD40 expression in SlMYC2-silenced plants to explore the upstream and downstream relationships between SlLBD40 and SlMYC2.

Here, we showed that SlLBD40, which belongs to subfamily II of the LBD family, was highly expressed in tomato roots and notably induced by PEG, salt, and methyl jasmonate (MeJA) treatments. Moreover, SlLBD40 was dependent on JA signaling and it might be downstream of SlMYC2. An overexpression and gene editing study showed that SlLBD40 functions as a negative regulator of drought tolerance. Knocking out SlLBD40 by CRISPR/Cas9 improved water-holding ability and enhanced drought tolerance in tomato.