The FBA Motif-Containing Protein NpFBA1 Causes Leaf Curling and Reduces Resistance to Black Shank Disease in Tobacco

Plant leaf morphology has a great impact on plant drought resistance, ornamental research and leaf yield. In this study, we identified a new gene in Nicotiana plumbaginifolia, NpFBA1, that causes leaf curl. The results show that the NpFBA1 protein contains only one unique F-box associated (FBA) domain and does not have an F-box conserved domain. Phylogenetic analysis placed this gene and other Nicotiana FBA genes on a separate branch, and the NpFBA1 protein localized to the nucleus and cytoplasm. The expression of NpFBA1 was induced by black shank pathogen (Phytophthora parasitica var. nicotianae) infection and treatment with salicylic acid (SA) and methyl jasmonate (MeJA). NpFBA1-overexpressing transgenic lines showed leaf curling and aging during the rosette phase. During the bolting period, the leaves were curly and rounded, and the plants were dwarfed. In addition, NpFBA1-overexpressing lines were more susceptible to disease than wild-type (WT) plants. Further studies revealed that overexpression of NpFBA1 significantly downregulated the expression of auxin response factors such as NtARF3 and the lignin synthesis genes NtPAL, NtC4H, NtCAD2, and NtCCR1 in the leaves. In conclusion, NpFBA1 may play a key role in regulating leaf development and the response to pathogen infection.


Introduction
The ubiquitin-proteasome system (UPS) is the predominant proteolytic pathway within eukaryotes. Ubiquitin is activated by covalent ligation to ubiquitin activating enzyme (E1), transferred to ubiquitin conjugating enzyme (E2) and added to ubiquitin ligase (E3), and then the ubiquitin labeled target protein is recognized and degraded by the 26S proteom [1]. The UPS regulates key physiological processes in plants, such as growth and development, hormone signaling, self-incompatibility, and resistance to pathogen invasion [2]. The F-box protein binds to the S-phase kinase-associated protein 1 (SKP1), cullin1 (CUL1) and RING-box protein (RBX) to form an E3 called the SCF complex that specifically recognizes the degradation substrate [3]. The selection of targeted substrates is often determined by the secondary domain of the F-box protein (FBP) [4]. The F-boxassociated (FBA) domain often appears as the C-terminal secondary domain of the F-box protein. In addition, secondary domains of the F-box include leucine zipper (LRR), WD and the effect of this gene on leaf morphological development and Phytophthora parasitica defense was initially examined.

Plant Materials
Seeds of the N. tabacum variety 'Honghuadajinyuan' (2n = 4x = 48) and N. plumbaginifolia (2n = 2x = 20) were provided by the National Medium Tobacco Intermediate Bank and later cultivated and harvested by the authors. Seeds of N. benthamiana were obtained from this laboratory. The seeds of N. tabacum and N. benthamiana were seeded directly in sterilized soil, dormant seeds of N. plumbaginifolia were placed on wet filter paper and treated with 100 µM gibberellic acid (GA3) [25], and the germinated seeds were then moved to sterilized soil. The tobacco materials were grown under the following growth chamber conditions: temperature of 24 • C and long-day photoperiod (16 h light/8 h dark). N. plumbaginifolia material was used for gene cloning and spatiotemporal expression, N. tabacum was used for transgene tests, and N. benthamiana was used for transient expression.

Culture of Pathogens and Production of Spore Suspension
P. parasitica var. nicotiana was provided by Professor Zhenlun Li, School of Resources and Environment of Southwest University, and originally collected from Fengjie County, Chongqing. Pathogens were grown on oat agar medium for 2 weeks, and the hyphae were scraped down and placed in 0.1% KNO 3 solution for 3 days. Hyphae were ground after storage at 4 • C for 30 min [26], the mycelium was later filtered with a mesh filter, the filtered spore suspension was placed under a microscope, and the number of sporocysts was recorded with a cell count plate. The sporocyst concentration of the sporulation suspension was finally adjusted to 1.4 × 10 3 sporangium·ml −1 for tobacco invasion experiments.

RNA Extraction and cDNA Synthesis
RNA from 2-month-old N. plumbaginifolia was extracted with an RNA extraction kit (DP441, TIANGEN, Beijing, China) and RNA mass and concentration were detected by 1% agarose gel electrophoresis and a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), respectively. First-strand cDNA was synthesized by using a PrimeScript TM RT reagent Kit with gDNA Eraser (RR047A, TAKARA, Shiga, Japan).

NpFBA1 Cloning and Sequence Analysis
Homologous cloning was performed using primers designed for the UTR of the N. tabacum FBA gene (XM_009598194), and then specific primers were designed (Supplementary Table S2) for the ORF region of the amplified sequences to eventually obtain the complete sequence of the N. plumbaginifolia FBA gene. All polymerase chain reaction (PCR) products were cloned into pMD19-T easy vector (TAKARA) for sequencing. The deduced amino acid sequences were aligned using the ClustalW program. A phylogenetic tree was constructed using MEGA 5.0 with the neighbor-joining method with 1000 bootstrap replicates with other sequences (Supplementary Table S3).

Subcellular Localization of NpFBA1 Proteins
The NpFBA1 sequences without a stop codon were linked to the pCAMBIA 2300-GFP vector using seamless cloning. Specific primers with restriction enzyme sites (BamHI) are listed in Supplementary Table S4. The constructed NpFBA1-GFP fusion protein and empty vector were then transformed into the Agrobacterium rhizomis strain (GV3101) and injected into the lower epidermis at OD = 0.1 with a needle-free syringe. After 48-72 h in the dark culture, the epidermis was removed to capture the fluorescence signal under an Observer DP80 fluorescence microscope (Olympus, Tokyo, Japan). Random fluorescent tobacco epidermal cells were captured with a charge-coupled device camera.

Hormone Treatment and Pathogen Infection
Two-month-old N. plumbaginifolia was treated with SA (2 mM) and methyl jasmonate (MeJA) (1 mM) hormones, with 0.1% alcohol treatment as a control. Each treatment was performed in triplicate, and the hormones were sprayed until the leaves bore water droplets. The leaves were taken at 0, 6, 12, 24, 48, and 72 h after treatment and frozen with liquid nitrogen.
N. plumbaginifolia leaves were taken for an isolated pathogen infection assay with three replicates. The leaf surface of the tobacco was punctured with five bound needles, 50 µL of the prepared spore suspension was placed on the wound of the leaves, and filter paper was placed over the wound to maintain moisture and prevent evaporation of the spore suspension. The leaves were taken at 0, 6,12,18,24,36,48 and 72 h and frozen with liquid nitrogen.

DNA Extraction from Transgenic and Wild Tobacco
The leaves of 2-month-old transgenic tobacco and wild-type (WT) tobacco were ground with liquid nitrogen, and then the genomic DNA of tobacco was extracted by modified CTAB method [28]. The detection method of DNA quality and concentration, refer to 2.1, and high-quality gDNA was diluted to 50 ng·µL −1 for PCR analyses. Primer pCAMBIA2300-NpFBA1 was used to identify transgenic plants.

Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)
qRT-PCR was conducted to detect changes in gene expression by using qTOWER 3 G (Analytik Jena, Jena, Germany). The PCR amplification procedure was as follows: 95 • C for 30 s, followed by 40 rounds of 95 • C for 20 s, 56 • C for 60 s, and a melting cycle of 65~95 • C. The system was a Novo Startfi SYBR qPCR SuperMix Plus 10 µL system (E096-01A, Novoprotein, Shanghai, China). The −∆∆CT method [29] was used to calculate the relative expression values, NpEF-1a and NtEF-1a were used as controls of N. plumbaginifolia and N. tabacum, respectively [30]. qRT-PCR analysis was repeated using three technical replicates. All the primers for qRT-PCR are shown in Supplementary Table S5.

Leaf Anatomy Observation
The tips of three mature leaves per plant were prepared for paraffin sections, cut into 0.5 cm 2 square blocks and then fixed with 24 h paraffin sections in FAA fixation solution with reference to previous methods [31]. Tobacco leaf thickness, fence tissue thickness, fence tissue density, sponge tissue thickness, and upper and lower epidermal cell thickness were observed under an Olympus fluorescence microscope, and each indicator of the leaves was measured by using the ruler in the photos taken by a charge-coupled device camera. Ten visual fields per leaf and three observations per visual field were used to calculate the average and deviation of each index. The cell tense ratio (CTR) and spongy ratio (SR) were calculated by the following formula: CTR% = fence tissue thickness/leaf thickness × 100; SR% = sponge tissue thickness/leaf thickness × 100.

Identification of the NpFBA1 Gene from Nicotiana plumbaginifolia
Based on homologous cloning techniques we isolated an FBA gene in N. plumbaginifolia, and named it NpFBA1 (Genbank accession number: OK216143). The alignment of NpFBA1 to the mRNA of the common tobacco FBA gene revealed that it has a base insertion at 651 bp, which results in a backward shift of stop codons and altered amino acid sequences, which may cause large functional differences. Amino acid sequence analysis showed that the NpFBA1 protein encodes 317 amino acids with a calculated molecular weight (MW) of 37.2 kDa and a deduced isoelectric point (PI) of 8.89, containing a conserved FBA domain ( Figure 1A). To reveal the evolution of NpFBA1, BLAST was performed with cloned NpFBA1 sequences and 16 homologues from other angiosperms. Phylogenetic trees of amino acid sequences of FBA gene in N. plumbaginifolia and other plants divided them into four groups based on genetic relationships ( Figure 1B). The NpFBA1 protein is located in the same clade (group II) as the FBA proteins of Nicotiana tabacum, Nicotiana tomentosiformis, and Nicotiana sylvestris, and domain analysis shows that this group contains only one FBA domain and no other domains. NpFBA1 is most closely related to the homologues in N. tabacum and N. tomentosiformis. The amino acid sequence of the FBA protein in N. tabacum was identical to that in N. tomentosiformis, which might indicate that the FBA gene of N. tabacum was derived from N. tomentosiformis. NpFBA1 is closely related to FBA genes in other Solanaceae plants, such as tomato and potato, and their FBA genes were placed in the same large clade (group I). The Hevea brasiliensis and Manihot esculenta FBA genes were located in the same clade (group III), and the FBA genes of the Camelina sativa, Tarenaya hassleriana, Arabidopsis thaliana, Brassica rapa, and Brassica napus were located in the same clade (group IV).
The tissue specificity of gene expression provides reference information for predicting and studying the biological functions of genes. We also examined the expression pattern of NpFBA1 in specific tissues, including roots, stems, leaves and flowers ( Figure 1C). We took the NpEF-1a gene of N. plumbaginifolia as the control gene, the expression level of NpFBA1 of flowers as unit 1, and the relative expression level was calculated by qPCR results using −∆∆CT method. The results showed that NpFBA1 was expressed in all of these tissues. The highest and lowest expression levels were observed in leaves and flowers, respectively. In addition, there were significant differences among roots, stems, leaves and flowers.

Subcellular Localization of NpFBA1
To determine the subcellular location of NpFBA1, we constructed a 35S::NpFBA1-GFP fusion controlled by the CaMV 35S promoter. The recombinant fusion vector and the control vector (35S::GFP) were transferred into the young leaves of tobacco (Nicotiana benthamiana) by Agrobacterium-mediated transformation. Then, the transgenic leaves were stained with 4, 6-diamidino-2-phenylindole (DAPI). As shown in Figure 2, the green fluorescence of the control was localized in both in the nucleus and cytoplasm, and the 35S::NpFBA1-GFP signal was also observed in the cytoplasm and nucleus. This indicates that the NpFBA1 protein localized to the nucleus and cytoplasm.

NpFBA1 Responds to Pathogen Infection and Exogenous Hormones
Based on previous studies, FBA genes may have a close relationship with disease resistance or sensitivity in plants. We treated N. plumbaginifolia with a spore suspension of P. parasitica and the 0.1% KNO 3 treatment as a control. qRT-PCR results showed that NpFBA1 was induced by pathogens within 0-72h, but this was a dynamic process. Compared with the control, NpFBA1 was not induced at 12h and 18h, and the induced expression was the highest at 72h, 28.5 times that of the control ( Figure 3A). The above results indicate that the NpFBA1 gene was responsive to infection by P. parasitica. We hypothesize that NpFBA1 plays a role in the plant defense response.
Plant hormones play crucial roles in the regulation of the expression of defense genes and the reactions against biotic and abiotic stresses [32]. SA and MeJA are important signaling molecules in plants that activate downstream defense-related genes [33]. To explore whether NpFBA1 is involved in plant defense pathways. N. plumbaginifolia was treated with SA and MeJA, using the expression of NpFBA1 in the untreated leaves as a control. N. plumbaginifolia treated with SA showed significantly higher NpFBA1 expression between 6-72 h, showing a tendency to rise, decrease, and rise again, with the highest expression after 72 h, 21 times that of the control ( Figure 3B). After MeJA treatment, NpFBA1 gene expression was suppressed at 6 h but induced at 48 and 72 h, with the highest expression at 48 h, 2.3 times that of the control ( Figure 3C). The above results indicate that the NpFBA1 gene responds to SA and MeJA hormone treatment and that NpFBA1 responds more strongly to SA than MeJA.

Functional Identification of the NpFBA1 Gene
To explore the function of NpFBA1, we constructed a 35S::NpFBA1 expression vector and then transformed it into common tobacco (N. tabacum). A total of 23 35S::NpFBA1 transgenic lines were obtained by kanamycin screening and PCR identification (Supplementary Figure S1). Gene function was studied using three transgenic lines with increasing expression ( Figure 4A).

Effects of the NpFBA1 Gene on Tobacco Growth and Development
To investigate the effect of the NpFBA1 gene on plant growth and development, we observed growth in WT and transgenic lines. During the rosette stage, most of the leaves of the transgenic tobacco were curled ( Figure 4B), and the leaves of transgenic lines were more mature than those of the WT, because we observed that NpFBA1#2 showed shedding of hairs, drooping of leaf tips, hardening of leaves, and reduction of sticky substance on tobacco surface ( Figure 4C). Curling of leaves, and plant dwarfing were also observed during the budding period of tobacco ( Figure 4D). Moreover, leaf curling and plant dwarfism became more evident with the expression of the three transgenic lines. The above phenomena indicate that NpFBA1 causes curling, aging and dwarfing of plant leaves. The leaf morphology of the transgenic plants also changed significantly during the bolting stage, with more rounded leaves than those of the WT ( Figure 5A,B). Leaf shape index (length/width) is an important parameter of leaf shape. The larger the leaf shape index of tobacco is, the longer and narrower the leaves are, while the smaller the leaf shape index is, the rounder the leaves are [34]. To verify whether the difference in leaf shape was significant, we measured the length and width of the fourth leaf from bottom to top and calculated the leaf shape index. The statistical results showed that the leaf shape index of the transgenic plants was significantly lower than that of the WT plants (Table 1), which indicated that the overexpression of NpFBA1 gene could lead to the change of tobacco leaf morphology. We speculated that was associated with the leaf curling phenotype.

Observation of the Leaf Anatomical Structure of WT and Overexpressed Plants
To further explore the cause of leaf curling of the transgenic lines, we took leaf tips from WT and transgenic lines for paraffin sections to observe the plant tissue structure of the leaf tips ( Figure 6). We further counted the thickness of fence tissue, sponge tissue, upper epidermis cells, lower epidermal cells, the spongy ratio (SR), cell tense ratio (CTR), and fence tissue density (Supplementary Table S1) in the four lines, but only a few structures had significant differences ( Table 2). Data analysis showed that several indicators of the overexpressing plants, including leaf thickness, superior epidermis thickness, sponge tissue thickness and fence tissue density, were significantly higher than WT, and the tissue structure tightness was lower compared with the control. Therefore, we speculate that the simultaneous increase in superior epidermal cell thickness and fence tissue tightness in overexpressing plants led to curling at the tip of tobacco leaves.

Pathogen Infection Status
Since NpFBA1 is highly expressed after infection by P. parasitica, we hypothesize that the NpFBA1 gene plays a role in the response to pathogen infection in plants. We used a spore suspension to inoculate leaves from plants overexpressing NpFBA1 and WT, and observed leaf damage. We found that during the tobacco rosette phase the transgenic line NpFBA1#17 was more susceptible to disease than the control after low concentrations of spore suspension treatment ( Figure 7A). Subsequently, we also performed in vitro pathogen infection experiments during the tobacco bolting stage. After 5 days of observation, we found that the transgenic lines were more susceptible than the WT plants. On the 5th day of infection, the spot area of the WT represented 2/3 of the entire leaf, while the whole leaf of the transgenic lines were almost completely damaged (Supplementary Figure S2). We further measured the disease spot area across periods and found that the transgenic lines showed notably higher values than the WT plants on both the fourth and fifth days after inoculation with the spore suspension ( Figure 7B). The above results suggest that the NpFBA1 gene negatively regulates plant resistance, consistent with the findings of previous studies of NpFBA1 family genes. Based on the dwarfism, leaf curling, and disease susceptibility phenotypes present in the transgenic lines, we hypothesized that leaf development-related genes (NtARF2, NtARF3 and NtPRTa) [35][36][37], the negative regulator gene of the GA pathway NtDALLE [38], and lignin synthesis genes (NtPAL, NtC4H, NtCAD2 and NtCCR1) [39], may be affected. We further examined the expression changes of the above genes in transgenic lines and WT plants (Figure 8). The results showed that the expression of auxin response factor NtARF2 regulating leaf maturation was significantly increased compared to that in WT plants; NtARF3 and NtPRTa genes involved in leaf morphogenesis were significantly down-regulated; the expression of NtDALLE, a negative regulator of the GA pathway, was significantly increased; and the expression of lignin synthesis-related genes (NtPAL, NtC4H, NtCAD2 and NtCCR1) was significantly reduced in transgenic lines. These results suggest that the NpFBA1 gene influences tobacco plant height and leaf development by affecting the auxin, gibberellin, and lignin signaling pathways.

Discussion
Leaf morphology has a great impact on plant drought resistance, ornamental research and leaf yield [40][41][42]. Although many genes [43,44] have been identified in plants that maintain proper leaf morphology, the genes directly controlling leaf shape, especially leaf curling, are unknown. Previous studies have shown that the 26S proteasomal degradation pathway mediated by the F-box protein is required for leaf development [45], and its secondary FBA domain is key for binding to downstream target proteins, but no functional studies of the FBA domain in tobacco have been performed. In this study, we isolated the gene NpFBA1 from N. plumbaginifolia to explore its potential role in leaf morphological development. Multiple alignment of NpFBA1 amino acid sequences with sequences from other species and phylogenetic analysis showed that NpFBA1 is in a separate clade from other tobacco FBA genes, mainly due to the absence of the F-box domain of NpFBA1 compared with other solanaceae plants. Subcellular localization experiments revealed that the NpFBA1 protein is localized to the nucleus and cytoplasm, consistent with previous findings in Arabidopsis [22]. Overexpression of the NpFBA1 gene leads to leaf curling, and plant dwarfing, providing a molecular basis for further studying the agronomic trait of plant leaf curling.
Auxin, a major regulator of cell proliferation, plays a crucial role in leaf morphogenesis and morphogenesis [46]. Thus, genetic modifications involving auxin homeostasis can lead to pleiotropic developmental defects, including leaf abnormalities. ARF is a key transcription factor regulating auxin signaling, and the ARF2 gene has been reported to regulate leaf senescence with floral organ shedding [35]. ARF3 physically interacts with the KANADI protein to form a functional complex essential for the maintenance of epidermal development and near-distal axis polarity in Arabidopsis [36]. In the NpFBA1overexpressing plants, leaf senescence related gene NtARF2 expression was significantly upregulated, we hypothesized that this might cause premature leaf senescence in transgenic plants. On the contrary, NtARF3 and Nt NtPRTa genes involved in leaf morphogenesis were significantly down-regulated, which may lead to leaf curl. In addition, we believe that significant downregulation of NtARF3 in transgenic tobacco leads to abnormal auxin production, which leads to abnormal development of leaf shape and plant height.
We found that the NpFBA1 gene was expressed in response to treatment with exogenous hormones (SA and MeJA) and P. parasitica, similar to the results for most F-box genes, such as BIG-24.1, TaPP2-A13, and TaFKOR23 [47][48][49]. Therefore, we hypothesized that this gene plays a role in plant-pathogen interactions. Interestingly, overexpression of the NpFBA1 gene did not enhance tobacco resistance to black shank disease, but instead attenuated its resistance, and these results were similar to those presented for the soybean NLL gene. The expression of GmNLL1 and GmNLL2 in soybean was significantly upregulated after infection with Phytophthora; however, overexpression of GmNLL1 and GmNLL2 in Arabidopsis did not enhance plant resistance to Phytophthora [50].
Notably, the regulatory immune regulatory pathway of the NpFBA1 gene in this study is likely different from that previously reported for CPR1/CPR30, and since the NpFBA1 gene has no conserved F-box domain, it likely does not trigger the degradation of the ubiquitin proteasome for the R protein. The plant susceptibility response mediated by the FBA gene is more likely to be caused by leaf aging and reduced lignin synthesis. Decreased activity of the reactive oxygen species scavenger superoxide dismutase (SOD) and accumulated reactive oxygen species in aging leaves can easily cause membrane lipid de-esterification, causing damage to the membrane structure and making pathogens more likely to invade plant cells. Lignin is a major component of the plant cell wall capable of protection against various biotic and abiotic stresses. It acts as a physical and chemical barrier to limit pathogen colonization and to limit pathogen growth in a variety of plants, and lignin has been used as a biochemical marker for detecting activated immunity [51]. phenylalanine ammonia-lyase (PAL), cinnamoyl CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and cinnamate 4-hydroxylase (C4H) are all key enzyme in the lignin synthesis pathway [39], and all four are severely downregulated in plants overexpressing NpFBA1, perhaps further leading to dwarfing and susceptibility in tobacco plants.
The current study of FBA gene-protein interactions has focused on the F-box motif at the N-terminus. The FBA gene family is often involved in the ubiquitin protease pathway through SKP1 binding to the F-box conserved domain, while target proteins at the C end of the F-box protein are very poorly reported. In Arabidopsis, some target proteins of FBA family genes have been reported, EIN3-BINDING F-BOX 1 (EBF1) regulate the plant ethylene pathway and promote plant frost resistance by targeting the proteins ETHYLENE INSENSITIVE3 (EIN3) and PHYTOCHROME-INTERACTING FACTOR 3 (PIF3) [52,53]. The Arabidopsis ABA-responsive FBA domain-containing protein 1 (AFBA1) gene combines with the transcription factors MYB44, TCP4 and TCP14 to regulate the ABA pathway [21]. The Arabidopsis CPR1/CPR30 protein interacts with the R proteins suppressor of npr1 constitutive1 (SNC1)and NBS-LRR protein (RPS2) and then degrades the R protein through the ubiquitin proteasome pathway to negatively regulate plant resistance. The diversity of FBA domain targets leads to the regulation of various aspects of the plant. Finding targets of the F-box protein is important for demonstrating its regulation of various aspects of plant growth and development. FBA motifs in F-box proteins function as identifying substrates and are essential for the normal function of F-box proteins, but the targets of most FBA genes and their functions are unknown.
Based on our results as well as the results of earlier studies, we conjectured that NpFBA1 and many uncharacterized F-box proteins containing functional FBA motifs share their target transcription repressors (TRs) involved in the negative regulation of various hormone response pathways, such as the auxin repressor Aux/IAA [54], and GA negative regulator DALLE [38]. On the one hand, the NpFBA1 protein competitively binds common targets downstream of the F-box-containing FBA protein so that this target protein cannot be recognized by FBA containing the F-box protein for UPS-mediated protein degradation. This suppresses the ubiquitin protease degradation pathway essential for plant growth and development, finally leading to abnormal plant growth and development; on the other hand, NpFBA1 may bind to unknown TRs and directly cause a decline in downstream lignin synthesis genes, resulting in reduced plant resistance to pathogens (Figure 9). However, the specific mechanisms by which NpFBA1 causes plant leaf curling remain to be investigated.

Conclusions
In this study, the NpFBA1 gene was located in the nucleus and cytoplasm and associated with leaf curling. Overexpression of NpFBA1 causes leaf curling and dwarfing of plants. Furthermore, we observed that overexpression of NpFBA1 led to tobacco susceptibility prone P. parasitica. Leaf anatomical analysis showed that superior epidermal cell thickness and fence tissue density were significantly higher than WT, and we speculate that this was one of the causes of leaf curl. Gene expression associated with lignin synthesis was significantly decreased in NpFBA1 overexpressing plants, leading to tobacco susceptibility to black shank disease. Although the NpFBA1 gene is strongly induced by P. parasitica and SA, it attenuates tobacco resistance to P. parasitica, which cannot be explained in our study. Our data provide insights into NpFBA1 regulation of tobacco leaf curl with pathogen defense, whereas the specific regulatory mechanisms remain to be further investigated.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/agronomy11122478/s1: Figure S1: Screening NpFBA1 transgenic lines; Figure S2: Leaf damage in the WT and transgenic lines at 0-5 days after P. parasitica infection; Table S1: Comparison of anatomical structure indexes of NpFBA1-overexpressing and WT plants; Table S2: Primers used for gene cloning; Table S3: Names and accession numbers used for phylogenetic analysis; Table S4: Primers used for vector construction; and Table S5: Primers used for quantitative real-time PCR (qRT-PCR).