The whole genomic sequences of pecan (Carya illinoinensis), walnut (Juglans regia), and hickory (C. cathyensis) are obtained from the Portal of Juglandaceae (Guo et al., 2020). Genomic data of ginkgo (Ginkgo biloba), amborella (Amborella trichopoda), waterlily (Nymphaea coloratar), soybean (Glycine max), grape (Vitis vinifera), rice (Oryza sativa), and Phalaenopsis (Phalaenopsis aphrodite) were obtained from the National Center for Biotechnology Information (NCBI) genome database. The protein sequences of A. thaliana were derived from TAIR¹.

A TLP keyword search of the NCBI for genes was conducted to find the coding sequence (CDS) of TLP, which were downloaded in FASTA format. The obtained TLP seed sequences were then searched against the previously downloaded whole-genome protein sequences using the BLASTX program (Camacho et al., 2009) (NCBI-BLAST 2.9.0 software, E-value of 10^–10) to look for similar homologous protein sequences of the candidate TLP genes in the 10 species. The seed files of TLP domains (PF00314) were downloaded from the Pfam database.² Candidate TLP protein sequences were further screened using the Hidden Markov Model algorithm of the HMMER software (Finn et al., 2011) and finally renamed as CcTLP1–40.

A portal, ExPASy,³ was used to identify and analyze the physicochemical properties of protein sequences (Gasteiger, 2003). Additionally, we predicted the location and transmembrane (TM) domains using Cell PLoc 2.0 (Chou and Shen, 2008) and TMHMM 2.0.⁴ The motif features of TLP sequences and signal peptides were analyzed with the MEME Suite and SignalP 4.0 (Petersen et al., 2011). Genome structure visualization was accomplished using the Gene Structure Display Server 2.0 online website (Hu et al., 2015) with a gff file. Promoter sequences were extracted using a pipeline of SAMtools and bedtools (Danecek et al., 2021). Then, the cis-acting elements were predicted using PlantCARE (Lescot, 2002).

MCSCAN software (Wang et al., 2012) was used to run the Pairwise Synteny Search program, with the whole-genome CDS of hickory and the gff file as input. The obtained anchor pairwise results were filtered with a 30-gene threshold for small fragment gene blocks. The identified TLP genes were searched in the final gene blocks.

MicroRNA (miRNA) is a class of non-coding single-stranded RNA molecules with a length of approximately 22 nucleotides that participate in the post-transcriptional gene expression regulation in plants and animals. The network profile between miRNAs and CcTLPs was further analyzed to better understand the miRNA regulation network of hickory TLP genes. The psRNATarget online server (Dai et al., 2018) was used to identify the miRNAs interacting with the hickory TLP genes. Then, the complex networks integrating miRNAs and TLP genes were visualized using Cytoscape (Shannon et al., 2003).

Multiple sequence alignment of TLP genes was achieved using MAFFT software (Katoh, 2002) under local pair mode, and the maxiterate parameter was set to 1,000. Then, TrimAL software (Capella-Gutierrez et al., 2009) was utilized to remove the spurious sequences and poorly aligned regions. The final alignment result was removed with more than 20% gaps, a similarity score lower than 0.001, and minimum conservation of 60%. The phylogenetic tree was built using the maximum likelihood method using IQTREE software (Nguyen et al., 2015). The s parameter was used to estimate the optimal substitution model automatically during the calculation process, and the bootstrap value was set to 1,000. JTT + I + G4 and JTT + R7 were the best-fit substitution models for hickory and green plants trees sorted by Bayesian information criterion (BIC) scores. The generated tree file was transfigured with the Evolview3 website (Subramanian et al., 2019).

In this experiment, the 6-month hickory seedlings growing in the Zhejiang Agriculture and Forestry University greenhouse (50 m, N30°23′, E119°72′) under a 16-h light/8-h dark photoperiod and at 22–25°C temperature were sprayed with 100 μM of SA, MeJA, and ETH until the first drop of liquid on the leaf surface. After treatments for 0, 2, 6, 12, 24, 48 h, 3–6 leaves from each plant were harvested, immediately frozen in liquid nitrogen, and stored at −80°C until use. This experiment was conducted with three independent biological replicates for each treatment.

The 2-year hickory seedlings were selected for B. dothidea inoculation in the Zhejiang Agriculture and Forestry University greenhouse (50 m, N30° 23′, E119° 72′). Before inoculation, B. dothidea was grown on potato dextrose agar (PDA) at 28°C in the dark. After 7 days, a 5-mm-diameter fungal agar plug was acquired using a punch and immediately placed on the surfaces of the wound site of the hickory stalk caused by the inoculating needle. In addition, a 5-mm-diameter empty PDA was used as control. The plug was removed after 2 days and the diameter of the disease areas was measured in six seedlings at 3, 4, 5, 6, 7, 9, 10, 11, 12, 14, and 16 days after inoculation (dpi) using rulers. The disease areas were collected at 0, 2, 7, and 16 dpi (Wang et al., 2020). All samples were immediately frozen in liquid nitrogen and then stored at −80°C until use. Each treatment contained three independent biological replicates.

Total RNA was extracted using a Quick RNA Isolation Kit (Huayueyang, China). cDNA was synthesized using the PrimeScript™ 1st strand cDNA Synthesis Kit (Takara, Japan) according to the manufacturer’s instructions. The gene-specific primers of TLPs and SA-synthesis and signaling-related genes were designed using the online software Primer 3.⁵ All primers used in this study are listed in Supplementary Table 1. The quantitative reverse transcription (qRT-PCR) was conducted using the SYBR Green Master Mix reagent (Applied Biosystems) and CFX 96 Real-Time system (Applied Biosystems), according to the manufacturer’s instructions. CcActin was used as an internal standard for normalization. The reaction procedure was 40 cycles with 95°C for 10 s and 55°C for 30 s. Formula 2^–ΔΔCT was applied to calculate the relative expression. Each sample was conducted with three biological replicates and three technical replicates in the RT-qPCR experiment. The relative expression level of CcTLP genes was calculated by the standard curve and then normalized by the CcActin expression level. R-package pheatmap was utilized to plot the expression heatmap of CcTLPs with SA, MeJA, and ETH treatments for 0, 2, 6, 12, 24, and 48 h.

The full-length CDS of CcTLP28, CcTLP29, CcTLP30, CcTLP31, CcTLP32, CcTLP33, CcTLP37, CcTLP38, and CcTLP39 were amplified with PCR. The gene-specific primers of the TLPs were designed using the Snapgene software and are listed in Supplementary Table 2. The PCR products were cloned into the binary vector 35s:GFP (modified from pCAMBIA 1300). The resulting plasmids with the correct sequence were introduced into Agrobacterium tumefaciens strains GV3101 and were cultured on the luria broth (LB) solid medium counting 50 μg/ml gentamicin (Geta), 50 μg/ml rifampicin (Rif), and 50 μg/ml kanamycins (Kana) at 28°C in the dark for 2 days. Then, a single colony was obtained, transformed into a liquid LB medium, and cultured at 28°C. After another 2 days, the cultures of A. tumefaciens (OD600 = 0.5–0.6) were centrifuged at 5,000 rpm at room temperature for 10 min and re-suspended in MMA buffer (10 mM MES, 10 mM MgCl₂, and 150 μM acetosyringone, pH = 5.6) to an OD600 of 1.0 and then incubated at room temperature in the darkness. After 2∼3 h, the suspension was injected into the 4-week-old tobacco (Nicotiana benthamiana) leaves. The plasma membrane marker (pm-rk) was used as a plasma membrane marker and was co-transformed with CcTLPs in tobacco (Nelson et al., 2007). After 2 days, green fluorescent protein (GFP) fluorescence was observed using laser confocal fluorescence microscopy (excitation: 488 nm; emission: 495–515 nm; LSM 800, Zeiss, Germany).

Statistical analyses were conducted using the one-way analysis of variance (ANOVA) procedure with SPSS (ANCOVA; SPSS26, SPSS Inc., Chicago, IL, United States). Significant differences among the groups were compared according to Duncan’s new multiple range test at p = 0.05.

After blasting and searching against the HMM seed model with whole-genome protein sequences, 40 TLP genes with typical thaumatin-like domains were found in C. cathayensis (Table 1). The number of TLP genes in 10 other green plants (G. biloba, A. trichopoda, N. coloratar, O. sativa, P. aphrodite, A. thaliana, G. max, V. vinifera, J. regia, and C. illinoinensis) are also listed in Table 1. Specifically, it has been shown that the amborella genome owns the least TLP genes. Compared with amborella, the number of TLP genes is almost two times in water lily belonging to the same Amborellales-Nymphaeales-Austrobaileyales (ANA) clade due to the Nymphaeaceae-specific whole-genome duplication (WGD) event (Zhang et al., 2020). Soybean and walnut genomes have the highest number of TLP genes, 62 and 66, respectively, almost three times than that of amborella, which may be attributed to that modern soybean was a diploid species from ancient tetraploid (Schmutz et al., 2010) and high quality of walnut genome (Marrano et al., 2020). From the information detected by the synteny analysis, four tandem duplications occurred in the distribution of TLP genes in hickory: CcTLP07 and CcTLP08 on the scaffold 18,053, CcTLP15, CcTLP16, and CcTLP17 on the scaffold 25,681, CcTLP21 and CcTLP22 on the scaffold 28,723, CcTLP29, CcTLP30, CcTLP31, CcTLP32, and CcTLP33 on the scaffold 54,619. Further synteny analysis with chromosome-level pecan genome showed these tandem duplicated TLP-located scaffolds originated from different chromosomes (Supplementary Figure 1). Five pairs of genes were revealed by gene synteny analysis to be duplicated from the WGD event, containing CcTLP15-CcTLP22, CcTLP16-CcTLP22, CcTLP17-CcTLP21, CcTLP18-CcTLP25, and CcTLP31-CcTLP38, which implied a potential influence of WGD event on CcTLP gene expansion.

Protein characteristics of hickory TLP gene family were further analyzed and presented in Table 2, such as Instability index (II), Aliphatic index (AI), Grand average of hydropathicity (GRAVY), and isoelectric point (pI). The II ranges from 26.65 (CcTLP29) to 73.31 (CcTLP08), and the AI values were between 52.1 (CcTLP35) and 83.67 (CcTLP27). According to GRAVY values, 35% (14 out of 40) of TLP genes code as hydrophobic, while others code as hydrophilic proteins. The pI values of 10 out of 40 CcTLP proteins reached values higher than seven, while the remaining ones reached values lower than seven. The vast majority of TLP genes were shown by sublocalization predication to be distributed in the cytoplasm. The exceptions were CcTLP14 and CcTLP18, located in the cell membrane/cytoplasm and vacuole, respectively. Finally, most of the genes contained transmembrane domains (Table 2). Notably, it was also found that some of these TLPs had N-signal peptides and transmembrane domains (TM) (Table 2).

The multi-sequence alignment was further performed and was shown in Figure 1. Most hickory TLP peptide sequences (35 out of 40) contain 16 cysteine residues and form eight stable disulfide bonds. However, CcTLP07, CcTLP09, and CcTLP27 lost five cysteines that only allowed them to encode type S (Small) TLPs with anti-fungal activity (Liu et al., 2010a). Moreover, it was observed that the CcTLP02 and CcTLP09 sequences were missing one and three amino acids, respectively. The REDDD structure from several genes has undergone varying degrees of mutation and deletion. For instance, CcTLP27 and CcTLP40 completely lost their REDDD structures, which may lead to an inability to maintain proper topologies (Liu et al., 2012). It was found that CcTLP24, CcTLP08, CcTLP29, CcTLP30, and CcTLP32 suffered 2–3 site mutations that may have affected their anti-microbial function. Notably, most of the hickory TLP sequences reversed the core domain sequence G-X-[GF]-X-C-X-T-[GA]-D-C-X(1,2)-G-X-(2,3)-C. Still, CcTLP27 missed part of the sequence: G-X-[GF]-X-C-X-T.

A total of 424 TLP genes were identified in 11 green plants to analyze the evolutionary relationship of TLP genes among green plants. The phylogenetic tree was distinctly divided into five subfamilies (Figure 2A). The green clade had the largest number of TLP genes (115, including 18 hickory TLP genes). Notably, this clade contains 37 gingko TLP genes and basal amborella TLP genes. This finding is an indication that the clade represents a complete evolutionary history. Blue, red, and purple clades had 117 (27.6%), 70 (16.5%), and 42 (9.91%) TLP genes, respectively. The red clade had 33 Juglandaceae species, consisting of 47.1% of the 70 TLP genes. This finding is a suggestion that the Juglandaceae-specific TLP gene family expansion was within the red clade.

A maximum-likelihood tree was constructed using 43 full-length TLP sequences (40 from hickory and three from Arabidopsis thaliana) to explore the further phylogenetic relationship among TLP genes in hickory (Figure 2B). The maximum-likelihood tree can be divided into three groups: group I (red line), group II (blue line), and group III (green line). The richest group III contained 22 TLP genes, including 19 CcTLP genes and three Arabidopsis TLP genes, which have been significantly studied. Within group III, CcTLP27 and CcTLP40 owned the longest clades, 2.6292 and 2.5801, respectively, indicating that these two genes were highly diverged. This result was consistent with the loss of REDDD in these two genes. The gene count in groups I and II was 12 and 8, and gene branch lengths in these two groups were nearly the same apart from CcTLP08. These results are suggestions that the genetic variation within group I and group II was low, and TLP gene functions were potentially similar.

Differences in the type and arrangement order of exons and introns could impact gene function. The gene structure of CcTLPs was analyzed and is shown in Figure 3B. Six CcTLP genes (CcTLP02, CcTLP35, CcTLP24, CcTLP40, CcTLP19, and CcTLP31) maintained only one exon, with no intron element within the genes. In the remaining, most of the genes (20 out of 35) contained two exons and one intron elements, 14 out of 35 genes had three exons and two introns, and remarkably CcTLP28 comprised four exons. Peptide sequence regions that have a significant impact on protein function or structure should be more conservative, which are called motifs. According to the motif analysis results, five motif patterns were found within 40 CcTLP genes (Figure 3C). Most of the CcTLP genes (82.5%) maintained the complete five motif patterns, while CcTLP09 and CcTLP40 lost one motif pattern, CcTLP02, CcTLP07, and CcTLP19 lost two motifs, and CcTLP08 and CcTLP27 only maintained two unbroken motifs.

Cis-acting elements are important short regions in the promoter sequence recognized by specific transcriptional factors, thereby regulating the activity of the promoter and the targeted gene expression. A batch of cis-acting elements was found by predicting the 2 kb CcTLP promoter sequences, such as light-responsive elements, hormonal regulation elements, and stress-related elements. Then, 65 emphasis elements related to stress-responsive, light-responsive, phytohormone-responsive, and plant-responsive were selected for classifying and accounting (Figure 4). Remarkably, the CcTLP11 promoter sequence contained 16 ARE elements involved in the antioxidative response. The CcTLP09 promoter sequence covered 12 ABRE elements, and 10 MYC elements participated in the phytohormone regulation process. The CcTLP genes were revealed by these results to be potentially involved in various life activity regulations, such as the antipathogen process, abiotic stress, and plant development.

In Figure 5, thirty-one miRNAs and 20 CcTLPs were predicted to be involved in the post-transcriptional process. Most of the CcTLPs (16 out of 29) were connected with one or two miRNAs. In contrast, CcTLP17, CcTLP26, CcTLP29, CcTLP34, and CcTLP38 were the most highly active genes to be influenced by four miRNAs. In addition, 24 out of 31 miRNAs targeted one or two TLP genes. Notably, miRNA4221 and miRNA5021 interacted with five TLP genes, suggesting that they had extensive roles in regulating the expression of TLP genes.

The SA and JA/ETH defense pathways are antagonistic, reflecting that enhanced resistance against biotrophs is often correlated with strengthened susceptibility to necrotrophs and vice versa. We selected nine CcTLP genes from the same clade with AT1G75040, AT1G75030, and AT1G18250 to quantify their expression levels in leaves after treatments with SA, MeJA, and ETH using qRT-PCR and to investigate the role of CcTLP genes, as demonstrated in Figure 6. It was shown that these most genes were all upregulated under SA treatment, especially CcTLP28, CcTLP29, CcTLP30, CcTLP33, and CcTLP38. However, CcTLP37, CcTLP38, and CcTLP39 were downregulated at all-time points under JA treatment. At the same time, CcTLP28 and CcTLP29 were significantly upregulated during all periods. Under ETH treatment, only two genes (CcTLP38 and CcTLP39) were found to be significantly upregulated. CcTLP29 and CcTLP30 had various changes in expression levels, being upregulated at some points and downregulated at others. These data are indications that CcTLP genes had different sensitivities to exogenous SA, JA, and ETH applications. It was suggested that the sequence of the CcTLP genes shared a high identity. Nonetheless, they had different expression patterns in response to the same hormone.

We further analyzed the changes in the previous nine CcTLP gene expressions to understand the function of TLP genes under infection conditions by B. dothidea. As shown in Figures 7A–C, B. dothidea caused dried and curly leaves, and the plant was led to death. When agar-containing mycelia was applied to the wound site, it became black, and the area enlarged was significantly different from control after 14 dpi. The diameters areas of the disease were broader after 3, 7, and 16 days of treatment (Figure 7D). Three CcTLP genes (CcTLP28, CcTLP29, and CcTLP30) were gradually upregulated with infection time. CcTLP31, CcTLP32, CcTLP33, and CcTLP38 could also be upregulated, and their expression levels were the highest after 2 dpi. Notably, CcTLP31 had an over 20-fold expression change. Based on these results, it is speculated that these genes were positively correlated with hickory resistance to B. dothidea (Figure 7E). In contrast, CcTLP37 and CcTLP39 were downregulated at the indicated time points compared with control (Figure 7E). This result is a suggestion that these CcTLP genes are negatively associated with hickory resistance to B. dothidea.

It has been reported that TLP proteins have antifungal functions. Subcellular localization analysis was further conducted to explore where they function. The GFP protein alone was present in the plasma membrane, cytoplasm, and nuclei, while the fluorescent signals of most CcTLPs-GFP were all found in the cytoplasm and plasma membrane (overlapped perfectly with the red fluorescence of the plasma membrane marker), as shown in Figure 8.