*2.5. Identification of Pray Proteins*

First, the CBL10 gene-specific protein sequence for the *Arabidopsis* plant was obtained from the official website of the *Arabidopsis* Information Resource database (ftp://ftp.arabidopsis.org; accessed on 21 February 2021). Then, the isolated protein sequence was used in a BLASTp analysis as a query against the proteomes of upland cotton, and the NAU assembly was used to identify the CBL10 homolog. The identified cotton CBL10 gene (*Gh\_D05G0440.1*) was later used in the Y2H system experiment to screen for its interacting proteins from the AD library.

#### *2.6. Construction of Yeast Two-Hybrid Library, Bait Cloning, and Auto Activation Analysis*

The Yeast Two-Hybrid (Y2H) fusion library of *Gossypium hirsutum* Marie-Galante leaves, stems, and roots under drought and salt conditions (pGADT7-library) was prepared by Oebiotech (Shanghai, China). The BD-*GhCBL10* bait plasmid was constructed as previously described [37]. In summary, the full length of the *GhCBL10* CDS was amplified by PCR, using the primers F-TGCATATGGCCATGGAGGCCGAATTC and R-TGCGGCCGCTGCAGGTCGAC GGATCC, and cloned at the pGBKT7 vector sites NCO1 and BamH1. It was crucial to confirm the transcriptional activation of the bait in the Y2HGold competent cell in the absence of a prey protein. We independently transformed the plasmids of bait, the negative control, and the positive control into Y2H Gold competent cells. The constructs were grown on different growth media, as described by Chen et al. [37], for three days. Table 1 presents the annotation of the negative and positive controls and the empty vector.


**Table 1.** The bait auto-activation and toxicity test sampling.

#### *2.7. cDNA Libraries Screening and Yeast Two-Hybrid Interaction Assay*

Yeast two-hybrid screening was conducted following the Oebiotech (Shanghai) mating protocol, as previously described. Briefly, we mated the bait strain (Y2HGold (pGBKT7- *GhCBL10*)) and the pGADT7-library plasmid, plated on the SD/–Ade/–His/–Leu/– Trp/Xα-gal/AbA (QDO/X/A) and SD/–Leu/–Trp/–His/X- α-gal/AbA (TDO/X/A) plates, and incubated the plates at 30 ◦C for five days [38]. We conducted colony PCR and sequencing using the T7 primer to determine the positive interaction and the duplicates. After sequencing, we used the BLASTn of the CottonFGD database to analyze the nucleotide sequence. We then co-transformed the potential positive prey identified with the pGBKT7- *GhCBL10* bait into Y2HGold competent cells. The CDS of CBL10 was cloned into the DNA-binding domain (BD) vector pGBKT7, while the CDSs of PRA1 B1, DSP8, and SAMS2 were cloned into the activation domain (AD) vector pGADT7, respectively, using the primers presented in Table S3. The generated transformants were grown on TDO/X/A and QDO/X/A plates and incubated at 30 ◦C until colonies appeared. PGBKT7-Lam and pGBKT7 (empty vector) denoted the negative control, while pGBKT7-53 denoted the positive control [38,39].

#### *2.8. Virus-Induced Gene Silencing of GhSAMS2 in G. hirsutum and Stress Treatments*

Tobacco rattle virus (pTRV) was used to elucidate *GhSAMS2* (*Gh\_A08G1067*) gene function with the RNAi technique [40]. VIGS TRV2:PDS, TRV2:00, TRV2:*GhSAMS2,* and WT plants were investigated under both drought and salt stress conditions. The CDS fragment of *Gh-SAMS2* was 1182 bp in length. The *GhSAMS2* cDNA was amplified using the specific primers F-TGCATATGGCCATGGAGGCCGAATTC and R-TGCGGCCGCTGCAGGTCGACGGATCC. Next, the PCR products were cloned into the *Xba*1 and *Xho*1 sites of the pTRV to generate pTRV:*GhSAMS2* [41]. Subsequently, recombinant DNA transformation into the LBA4404 bacteria strain (*Agrobacterium tumefaciens*) was conducted as previously described [42]. The LBA4404 strain containing the pTRV2-PDS, pTRV1, pTRV2-*Gh\_A08G1067*, and pTRV2 vectors was cultured in a shaking incubator at 28 ◦C in the Luria-Bertani (LB) liquid medium, with freshly prepared 10 mM 2-(N-morpholino)- ethane sulfonic acid (MES) added in. Kanamycin and rifampicin antibiotics were first added to the LB medium. Then, the cultures were put in the shaking incubator overnight, as previously prescribed [43]. This was followed by the centrifugation of the cultures for 10 min at 8000 rpm after the OD had been determined at 1.5, and the cells then were re-suspended into the infiltration buffer containing 200 μM of acetosyringone (As), 10 mM of magnesium chloride, and 10 mM of MES to a final OD600 = 1.5. To obtain the final infiltration medium, the pTRV1 re-suspension was mixed with pTRV2-PDS, pTRV2-*GhSAMS2,* and pTRV2, separately, at a ratio of 1:1 before the seedlings were infiltrated by the infusion, as previously described [44]. The functional analysis experiment via VIGS involved the inoculation of 60 plants with the TRV:*GhSAMS2* and TRV: PDS inoculum, respectively. The empty vector (TRV2:00) was inoculated into 60 other plants to represent the wild type. Then, 60 other plants were left to grow without any inoculum, serving as the control in this experiment. When the plants reached the three-leaf-stage, the Hoagland nutrient solution medium into which they had been transplanted was treated with freshly prepared solutions of 17% of glycol PEG-6000 and 250 mM of sodium chloride compounds to simulate drought and salt stress, respectively [45]. The duration of each stress was 48 h. After the stress exposure, the healthy tissues of the stem, root, and leaf were collected from ten plants of each category in triplicate for RNA extraction and physiological and biochemical analyses.

#### *2.9. Measurement of the Physiological and Morphological Parameters*

The morphological and physiological parameters were equally determined to help assess the extent of susceptibility between the silenced and non-silenced plants under drought and salt stress conditions. Plant height (PH), root length (RL), shoot fresh weight (SFW), root fresh weight (RFW), relative leaf water content (RLWC), cell membrane stability (CMS), chlorophyll content (SPAD/Chlo), and excised leaf water loss (ELWL) were measured. The relative leaf water loss, cell membrane stability through ion leakage, and chlorophyll content were determined, as described previously [46]. Excised leaf water loss was determined by first weighing the collected fresh leaf samples immediately after harvesting to note the initial leaf weight in grams. After the leaf sample had lasted for 24 h on the bench at room temperature, the second weight measurement was taken and recorded as wilted weight (WW). Finally, the third measurement was taken and recorded as dry weight (DW) after the leaf sample had stayed inside an oven (50 ◦C) for four days. To calculate the ELWL, the formula below was applied.

$$\text{ELWL} = \left\{ \frac{\text{FW} - \text{WW}}{\text{DW}} \right\}.$$

Regarding the relative leaf water content and fresh weight (FW), the leaf samples were placed into dd H2O at room temperature for 24 h using tissue paper; they were then dried on both surfaces before being weighed again to obtain the saturated weight (SW). Finally, the dry weight (DW) was measured and recorded after the leaf samples had stayed inside an oven at 50 ◦C for four days. The formula applied in the calculation of RLWC was:

$$\text{RLWC} = \left\{ \frac{\text{FW} - \text{DW}}{\text{SW} - \text{DW}} \right\} \times 100$$

Ion leakage in the plant tissues, which is also referred to as cell membrane stability (CMS), was assessed using the fresh leaf tissues. First, the plant electrolyte was quantified in the process of determining cell membrane stability, as previously described [47]. Then, plastic cylindrical tubes filled with 5 mL of dd H2O and kept in the dark for 24 h were used to harbor leaf samples weighing 0.5 g each. Two electrical conductivities were measured per sample, the first one being measured after the 24 h dark period stage (T1), while the second one was conducted after the leaves had been boiled in a water bath at 99 ◦C for 30 min and cooled to room temperature (T2). The CMS was calculated using the following formula [48]:

$$\text{CMS} = \left[ \left( 1 - \frac{\text{TI}}{\text{T2}} \right) / \left( 1 - \frac{\text{C1}}{\text{C2}} \right) \right] \times 100$$

where C is the electrical conductivity of dd H2O.
