Genome-wide characterization and expression of the TLP gene family associated with Colletotrichum gloeosporioides inoculation in Fragaria × ananassa

Introduction

Thaumatin-like proteins (TLPs) are important members of the highly complex gene family pathogenesis-related protein group 5 (PR5). They are highly homologous with sweet-tasting thaumatin protein produced by Thaumatococcus daniellii fruit (Wel & Loeve, 1972; Velazhahan, Datta & Muthukrishnan, 1999; Christensen et al., 2002; Loon, Rep & Pieterse, 2006). TLPs are abundant and highly diversified in plants (Loon, Rep & Pieterse, 2006; O’Leary, Poulis & von Aderkas, 2007; Liu, Zamani & Ekramoddoullah, 2010). According to a survey conducted in the UniProt database (https://www.uniprot.org/uniprot/?query=Thaumatin-like+proteins&sort=score), 1,816 TLPs (1,720 angiosperms, 82 gymnosperms, seven bryophytes, and seven algae species) from 187 different species have been identified (Faillace et al., 2019; Jesús-Pires et al., 2020). A great number of isoforms activated by biotic or abiotic stresses and multiple rounds of distinct gene duplication events were observed across the TLP gene family’s expansion and evolution in plants, suggesting that this superfamily has a complex pattern of molecular evolution (Liu, Zamani & Ekramoddoullah, 2010; Cao et al., 2016). Previous studies have indicated that plant TLPs consist of approximately 200 amino acids, have a molecular weight of 21–26 kD (Velazhahan, Datta & Muthukrishnan, 1999), and contain 10 to 16 conserved motifs (Hulo et al., 2008). These proteins exhibit a series of responses to biotic and abiotic stress factors in plants, such as pathogen invasion, drought, wounding, freezing, and salinity (Petre et al., 2011). They also play roles in a variety of physiological and developmental processes, including organ formation, fruit ripening, and seed germination (Salzman et al., 1998; Seo et al., 2008).

The octoploid cultivated strawberry (Fragaria × ananassa, 2n = 8x = 56) is an economically important perennial horticultural crop (FAO, IFAD, UNICEF, WFP, WHO, 2017) that is widely cultivated in China, but the main cultivar is ‘Benihoppe’ from Japan. ‘Benihoppe’ is susceptible to a range of diseases including anthracnose, which is one of the most destructive fungal diseases that results in considerable losses in strawberry production (Hammerschlag et al., 2006; Dean et al., 2012), especially at the seedling stage and early stages following transplanting. On the east coast of China, anthracnose in strawberry is mainly caused by the fungus Colletotrichum gloeosporioides (Zhang et al., 2017), which can infect all aerial plant parts, and the most severe symptoms are dwarf-stem and foliar lesions.

Pathogenesis-related (PR) proteins, including TLPs, exhibit significant antifungal activity in plants (Velazhahan, Datta & Muthukrishnan, 1999). TLP gene expression is responsive to infection by a variety of fungal pathogens, such as Colletotrichum, Podosphaera, Phytophthora, Fusarium, and Neurospora (Woloshuk et al., 1991; Narasimhan et al., 2003; Liu, Zamani & Ekramoddoullah, 2010; Rather et al., 2015). TLPs play multiple roles in inhibiting hyphal growth and spore germination of various pathogenic fungi (Roberts & Selitrennikoff, 1990; Woloshuk et al., 1991; De Freitas et al., 2011), binding β-1,3-glucans to destroy the stability of fungal membranes (Grenier, Potvin & Asselin, 2000; Osmond et al., 2001; Zareie, Melanson & Murphy, 2002), and stimulating plant defensive responses against pathogenic fungi to prevent fungal damage or reduce disease symptoms. Because of their effective antifungal activity, TLP genes are promising candidates for improving plant disease resistance. Overexpression of defense-related TLP genes in transgenic Arabidopsis (Rout, Nanda & Joshi, 2016), rice (Datta et al., 1999), tobacco (Singh et al., 2013), grape (He et al., 2017), wheat (Mackintosh et al., 2007), and potato (Acharya et al., 2013) significantly enhanced plant resistance to fungal diseases.

Resistance to a variety of pathogens has been a key breeding purpose in the development of commercial strawberry cultivars. Research on plant defensive responses against fungal pathogens indicate that TLPs are promising candidates for developing better anthracnose resistance in strawberry. Although the TLP gene has been studied in a variety of plants, the composition of the TLP gene family members in octoploid strawberry and their potential defense response to the invasion of pathogenic fungi, particularly C. gloeosporioides, remain largely unexplored. Additionally, many studies on strawberry anthracnose have focused on fungicides used for disease control, but not the molecular resistance mechanisms and engineered plant disease resistance of the octoploid strawberry.

This study aimed to identify the TLP gene family members in octoploid cultivated strawberry (F. × ananassa), and examine their transcription and expression characteristics during pathogenic fungi infection. The purpose of this study was to expand our understanding of the function of strawberry TLP genes in defense responses against C. gloeosporioides.

Materials and Methods

Results

Phylogenetic relationships of TLPs in major plant species

To investigate the evolutionary relationship of the TLP gene families, 10 representative Arabidopsis thaliana and Oryza sativa TLPs sequences were used as reference sequences together with F. × ananassa and F. vesca TLPs in the phylogenetic analysis. Similar to the phylogenic results of Arabidopsis and Oryza TLPs (Shatters et al., 2006), the 76 FaTLPs were also classified into 10 phylogenetic groups, Groups I to X (Fig. 1A). A maximum number (16) of FaTLPs were clustered in Group VIII, followed by Group V (15), and these two groups accounted for almost half of the FaTLPs. Groups III and IX contained the least amount of TLP family genes with two genes each. Similarly, most of the F. vesca TLPs were concentrated in Groups VIII and V, and their distribution was more uneven than the FaTLPs.

Conserved motifs of TLP genes

The diversity of the motif compositions of TLP genes in F. × ananassa was assessed using MEME software, and a total of 15 conserved motifs were obtained. The distribution of these 15 motifs in the TLPs are shown in Fig. 2. FaTLP10 and FaTLP68 contained all 15 conserved motifs. In addition, four genes (FaTLP55, FaTLP56, FaTLP59, and FaTLP62) contained 14 motifs but did not include motif11. Motif1, 2, 3, 4, 5, 6, 7, 10, 11, and 15 were found in most of the FaTLP genes. Furthermore, conserved motif6 was widely distributed across all 76 FaTLP genes. Motif2 and motif7 were absent only in one gene (FaTLP5), and motif3 was not found in two genes (FaTLP9 and FaTLP33). These 10 conserved motifs were also common across all groups (Group I to X). Moreover, some members of Group V (FaTLP10, FaTLP11, FaTLP55, FaTLP56, FaTLP58, FaTLP59, FaTLP62, FaTLP64, and FaTLP68) shared several unique motifs, namely motif8, 9, 12, 13, and 14. These results suggested that the TLP genes in each group shared several unique motifs and may have certain functional similarities. Moreover, these motifs were relatively conserved, which is why they may be used as markers for the identification of TLP genes and important functional components of the TLP gene family.

qRT-PCR analysis of TLP genes in response to C. gloeosporioides infection

To further understand the roles of TLP genes in strawberry, we investigated the expression profiles of FaTLP genes in F. × ananassa. The qRT-PCR results of these TLP genes in different resistant strawberry cultivars after infection with the fungal pathogen showed a wide range of expression responses (Fig. 3B, File S1). The induction expression of FaTLP68 was significant in the resistant cultivar (‘Kaorino’) at 12 hpi, although it decreased over time, whereas the upregulation of FaTLP68 was detected at 24 hpi in the susceptible cultivar (‘Benihoppe’), and its expression peak appeared later than that of the resistant cultivar. FaTLP40 and FaTLP41 were also highly induced in both strawberry cultivars but there were two expression peaks. This induction increased over time and reached its first peak at 24 hpi and the second peak at 4 d post-inoculation (dpi) in the resistant cultivar, whereas the susceptible cultivar had its first expression peaks of FaTLP40 and FaTLP41 at 48 hpi. The expression of FaTLP62 was considerably upregulated at 24 hpi only in the susceptible cultivar. These results showed a trend similar to the one observed using RNA-seq. Nevertheless, a gradual downregulation was observed for FaTLP43 and FaTLP75 in ‘Kaorino’ after pathogen infection, which then significantly increased at 12 hpi and 48 hpi, respectively, followed by a decrease until 7 dpi. This was not entirely consistent with the results of the transcriptome analysis, indicating that FaTLP43 and FaTLP75 were downregulated at 24 hpi. However, in the susceptible cultivar, the expression patterns of FaTLP43 and FaTLP75 were similar during the initial stage after inoculation but the maximum peak occurred at 4 dpi (1 day after disease symptoms were visible to the naked eye; Fig. 3A). Overall, the qRT-PCR analysis indicated a clear upregulation of the six abovementioned TLP genes following C. gloeosporioides infection in the octoploid strawberry.

Discussion

TLPs are a PR protein family that plays a key role in plant defense. In this study, 76 TLP gene members were identified in F. × ananassa, and their characteristics, phylogenetic relationships, motif organization, and chromosomal location were investigated. Using reference sequences from Arabidopsis and Oryza, F. × ananassa TLP genes were clustered into 10 paraphyletic groups, although the distribution of the TLP genes in each group was not uniform. Phylogenetic studies using Arabidopsis, Oryza (Shatters et al., 2006), Populus (Zhao & Su, 2010) and barley (Iqbal et al., 2020) species found that TLPs were also unevenly distributed across the groups, whereas the largest groups contained 11 (36.67%), 5 (23.81), 25 (45.45%), and 7 (36.84%) TLPs, respectively. Previous studies hypothesized that those clades containing the largest gene members would be more active against pathogens or have other exogenous stimuli and more rapid adaptive evolution (Zhao & Su, 2010). In this study, Groups V and VIII were the largest gene clades in our paraphyletic results. Our reference TLP sequence (AT4G11650) from Arabidopsis, annotated as an osmotin-like gene ATOSM34, was found in Group V. Three other PR TLP genes from Arabidopsis (At5g24620) and Oryza (Os07g23470 and Os08g40600) involved in defense response to pathogens (Ascencio-Ibáñez et al., 2008; Liu, Zamani & Ekramoddoullah, 2010) were also clustered in Group V and Group VIII, although their exact functions have not been verified. Osmotin is a TLP belonging to the PR5 family that was originally regarded as a salt-induced protein (Singh et al., 1989), and its antifungal mechanism of action has been studied in detail (Xu et al., 1994; Ibeas et al., 2001; Salzman et al., 2004). ATOSM34 has been reported as a key Arabidopsis gene involved in the defense response to biotic and abiotic stress (Capelli et al., 1997; Mukherjee et al., 2010; Vibhuti et al., 2016; Park & Kim, 2021). We used transcriptome data to identify the six genes encoding TLP proteins associated with plant responses to C. gloeosporioides infection, FaTLP40, FaTLP41, FaTLP43, FaTLP62, FaTLP68, and FaTLP75, which were also clustered in Group V (FaTLP40, FaTLP41, FaTLP62, and FaTLP68) and Group VIII (FaTLP43 and FaTLP75), respectively. These FaTLP genes clustered in the same clade as the reference genes, which have anti-fungal functions, indicating that the TLP genes belonging to the same phylogenetic group may have certain functional similarities.

Further qRT-PCR analysis verified that five of the TLP genes were up-regulated in resistant strawberry cultivars upon C. gloeosporioides infection (Fig. 3). Upregulation or overexpression of TLP genes often results in enhanced antifungal activity against several different pathogenic fungi (Datta et al., 1999; Fagoaga et al., 2001; Kalpana et al., 2006). For example, overexpression of barley TLP-1 in transgenic wheat lines improved pathogen resistance to Fusarium graminearum (Mackintosh et al., 2007). Similarly, increased TLP gene expression in transgenic tobacco plants enhanced resistance to Pythium aphanidermatum and Rhizoctonia solani (Rajam et al., 2007), while overexpression of the TLP gene VaTLP improved downy mildew resistance in Vitis vinifera (He et al., 2017). In turn, significant upregulation of FaPR5-1 and FaPR5-2 was observed in the salicylic acid-primed defense response of octoploid strawberry to Podosphaera aphanis (Feng et al., 2020). Ultimately, these results suggest that FaTLP40, FaTLP41, FaTLP43, FaTLP68, and FaTLP75 may have potential functions in plant resistance responses to C. gloeosporioides.

Our findings demonstrated an initiation of transcriptional responses in strawberry leaves during C. gloeosporioides invasion for some FaTLP genes. Additionally, the expression patterns of these genes revealed that the two different resistant strawberry cultivars differed in their temporal defense responses. FaTLP43 and FaTLP75 activated defense responses much faster in ‘Kaorino’ (<48 hpi) than in the susceptible cultivar ‘Benihoppe’ (4 dpi). Therefore, we hypothesized that during C. gloeosporioides inoculation in strawberry leaves, the spread of the fungus was suppressed because of a early defense response in the resistant cultivar while the pathogen spread out of control in the susceptible cultivar due to the delayed defense response. Similar results were also reported in the response of strawberries to Verticillium dahliae (Besbes, Habegger & Schwab, 2019) and Podosphaera aphanis (Feng et al., 2020) infection, which supports our hypothesis. The early activated defense mechanism of these TLP genes in resistant cultivar such as ‘Kaorino’ needs to be further studied. Our results provide a new strategy for developing anthracnose-resistant strawberry cultivars in the future by promoting early response TLP gene expression with antifungal activity in susceptible cultivars.

Conclusion

In this study, we performed genome-wide identification and characterization of TLPs in octoploid strawberry. A total of 76 TLP genes (FaTLP1–76) were identified using genome-wide screening. Comparative phylogenetic analysis divided the TLPs into 10 groups and the functions of TLPs in F. × ananassa were analyzed. Our qRT-PCR analysis indicated a clear upregulation of five TLP genes in resistant strawberry leaves infected with C. gloeosporioides. Furthermore, our results showed differences in TLP gene expression patterns between two different resistant strawberry cultivars. We concluded that the TLPs’ early activated defense to pathogenic fungi might be a reason why the resistant strawberry cultivar ‘Kaorino’ showed greater anthracnose resistance than the susceptible cultivar ‘Benihoppe’. This study lays the foundation for further exploration of the antifungal function of TLP genes in F. × ananassa, and provides new strategies for improving strawberry resistance to anthracnose through genetic engineering.

Supplemental Information