Unraveling NPR-like Family Genes in Fragaria spp. Facilitated to Identify Putative NPR1 and NPR3/4 Orthologues Participating in Strawberry- Colletotrichum fructicola Interaction

Abstract

The salicylic acid receptor NPR1 (nonexpressor of pathogenesis-related genes) and its paralogues NPR3 and NPR4 are master regulators of plant immunity. Commercial strawberry (Fragaria × ananassa) is a highly valued crop vulnerable to various pathogens. Historic confusions regarding the identity of NPR-like genes have hindered research in strawberry resistance. In this study, the comprehensive identification and phylogenic analysis unraveled this family, harboring 6, 6, 5, and 23 members in F. vesca, F. viridis, F. iinumae, and F. × ananassa, respectively. These genes were clustered into three clades, with each diploid member matching three to five homoalleles in F. × ananassa. Despite the high conservation in terms of gene structure, protein module, and functional residues/motifs/domains, substantial divergence was observed, hinting strawberry NPR proteins probably function in ways somewhat different from Arabidopsis. RT-PCR and RNAseq analysis evidenced the transcriptional responses of FveNPR1 and FxaNPR1a to Colletotrichum fructicola. Extended expression analysis for strawberry NPR-likes helped to us understand how strawberry orchestrate the NPRs-centered defense system against C. fructicola. The cThe current work supports that FveNPR1 and FxaNPR1a, as well as FveNPR31 and FxaNPR31a-c, were putative functional orthologues of AtNPR1 and AtNPR3/4, respectively. These findings set a solid basis for the molecular dissection of biological functions of strawberry NPR-like genes for improving disease resistance.

Keywords: NPR-like family; anthracnose; expression profile; phylogeny; strawberry.

Figure 1

The phylogenetic relationships of NONEXPRESSOR OF PATHOGENESIS-RELATED GENES 1 (NPR1)-like proteins from Arabidopsis thaliana, five diploid strawberries, and the octoploid strawberry. The tree was clustered into three clades (I, II, and III) shaded with different colors. The length of branches indicates the relative phylogenetic relationship, and the bootstrap values near branches for confidence. Geometries with different shapes and colors are used to symbolize different NPR members, with a star for Arabidopsis, and cycles in red, yellow, green, purple, pink, and gray for Fragaria vesca, F. iinumae, F. viridis, F. nipponica, F. nilgerrensis, and F. × ananassa, respectively.

Figure 2

The comparative organization of exons and introns in 48 strawberry NPR1-like genes and 6 AtNPRs. The structure was produced with the gene structure display server (GSDS) at http://gsds.cbi.pku.edu.cn/Chinese.php (accessed on 27 February 2022). The blue and the yellow rectangles represent the un-translated regions (UTR) and the coding sequences (CDS), respectively. The neighbor-joining tree of the NPR1-like genes was generated with MUSCLE clustering by MEGA7.0 for CDS nucleotide sequences.

Figure 3

The comparative protein domain module of six AtNPRs and 48 strawberry NPR1-like sequences. The deduced protein sequences in clade I (A), clade II (B), and clade III (C). The locations of the conserved BTB/POZ domain, the Ankyrin repeats (Ank_2 or Ank_5), and the NPR1/NIM-like defense protein C-terminal region (NPR1_like_C) were revealed via using the web CD Search Tool at NCBI.

Figure 4

The chromosomal distributions of NPR1-like genes in Fragaria vesca (A) and F. × ananassa (B). The identity of each chromosome (of certain sub-genome) is shown at the top. The scale ruler at the left side indicates the physical distance of chromosomes in megabases (Mb). The location site and the transcriptional direction for each strawberry NPR1-like locus are marked as triangle.

Figure 5

The predicted cis-elements in the promoter regions of NPR-like genes in Fragaria vesca and F. × ananassa. The promoter sequences (−2500 bp upstream of the starting code ATG) of 48 strawberry NPR-like genes were analyzed by PlantCare. The geometries in different colors and shapes indicate elements involved in different processes, with cycles for hormone-responsive, filled rectangles for stress-responsive, and rectangles with gradient color for the others.

Figure 6

The expression responses of strawberry NPR-like genes upon Colletotrichum fructicola invasion in diploid ‘Hawaii4’ and octoploid cultivars ‘Camarosa’ and ‘Benihoppe’. (A) The typical symptoms induced by C. fructicola on wounded detached strawberry leaf blades at 25 °C were photographed at 4 and 5 dpi. The left and right side of each leaf blade (adaxial side up) was inoculated with two 10-μL droplets of sterile water with 0.01% (v/v) Tween 20 (Mock, M) and conidia suspension (2 × 10⁶ per mL, C. f), respectively. Scale bar, 1 cm. (B) The semi-quantitative RT-PCR analysis of strawberry NPR-like genes. The third, fourth, and fifth compound leaves of sprayed inoculated plants (C. f) or mock-treated (M) were harvested at different hours post inoculation (hpi). The PCR cycle numbers are 40 and 28 for NPR1-likes and the internal control EF1α, respectively. In amplification for FveNPR31, -32a, -32b, and -33, 0.2-μL original cDNAs was used as a template, while for NPR1 and -5 in both diploid and octoploid strawberry, 1-μL original cDNAs was used.

Figure 7

A quantitative RT-PCR analysis of strawberry NPR3-like genes upon C. fructicola invasion in diploid ‘Hawaii4’ and octoploid ‘Benihoppe’. The same RNA samples in Figure 6 were analyzed. The same primer pairs for all NPR3-like genes except for NPR32b were used in F. vesca ‘Hawaii4’ and F. × ananassa ‘Benihoppe’. FvePR10a (FvH4_4g19120) was used as a marker gene of SA-depending defense signaling pathway. The primers and corresponding target alleles amplified were shown in Supplementary Table S6. The gene name followed with ‘-s’ indicates multiple alleles detected in ‘Benihoppe’. The relative transcript levels of NPR3-like genes were normalized with two reference genes EF1α and GAPDH2 [49] and reported as the mean of three biological replicates ± SE. The asterisks indicate significant differences based on a Student t-test analysis (*, p < 0.05).

Figure 8

The protein–protein interaction networks proposed for the strawberry FveNPR1-like proteins in STRING (https://cn.string-db.org, accessed on 26 February 2022). The edges in different colors represent the predicted interaction relationships according to different methods. The detailed information for all potential partners is shown in Figure 9 and Figure S3.

Figure 9

RNA-seq data showing the differential expression of genes coding proteins potentially directly or indirectly interacting with NPR1-like proteins (A) and the other known members in SA signaling (B) in strawberry during the C. fructicola invasion. The heatmap was generated using RPKM (reads per kilobase per million mapped reads) values normalized via Log2-transformation for each transcript in a moderate susceptible strawberry cv. ‘Jiuxiang’ mock-treated or infected by C. fructicola at 24, 72, or 96 hpi. The black star symbols indicate NPR family members. Strawberry materials and C. fructicola inoculation conditions for RNA-seq data generation have been reported previously [35].

Figure 10

A hypothetical model depicting the transcriptional events related to NPR1- and NPR31-mediated defenses in susceptible strawberry upon invasion with a hemibiotrophic fungal pathogen Colletotrichum fructicola. (A) At 6 hpi early stage, the strawberry cell accumulates a relatively low or basal level of SA (red cycles). There is no monomeric NPR1 protein (ellipse), and it exists in cytoplasm as a large oligomer or forming heterodimer with the negative regulator NPR31. In the nucleus, the transcription of NPR1 is suppressed, while NPR31 transcription has been restored to a steady state after a transient induction. Simultaneously, the expression of pathogenesis-related gene PR1, as well as SA synthesis genes ICS and PAL, show a similar dynamic pattern to that of NPR31. All events indicate a fast quenching of SA-dependent resistance, resulting in effector (small brown shapes)-triggered susceptibility (ETS). (B) At the 96 hpi late necrotrophic stage, the strawberry cell contains elevated SA due to the increased expression of PAL1/2. SA-dependent resistance is partially activated after NPR1 binds SA. Enhanced NPR31 negatively regulates SA-related immunity. Meanwhile, SA-dependent resistance is antagonized by the disinhibition of MeJA-mediated defense and the activation of ethylene-related defense, which is beneficial for strawberry defending the pathogen with a necrotrophic life at 96 hpi. There exists monomeric NPR1 and NPR31 in the nucleus, which directly target TGA transcriptional factors to regulate the expression of PR genes. Members are in red characters for up-regulated and in blue for down-regulated.

Figure 10

A hypothetical model depicting the transcriptional events related to NPR1- and NPR31-mediated defenses in susceptible strawberry upon invasion with a hemibiotrophic fungal pathogen Colletotrichum fructicola. (A) At 6 hpi early stage, the strawberry cell accumulates a relatively low or basal level of SA (red cycles). There is no monomeric NPR1 protein (ellipse), and it exists in cytoplasm as a large oligomer or forming heterodimer with the negative regulator NPR31. In the nucleus, the transcription of NPR1 is suppressed, while NPR31 transcription has been restored to a steady state after a transient induction. Simultaneously, the expression of pathogenesis-related gene PR1, as well as SA synthesis genes ICS and PAL, show a similar dynamic pattern to that of NPR31. All events indicate a fast quenching of SA-dependent resistance, resulting in effector (small brown shapes)-triggered susceptibility (ETS). (B) At the 96 hpi late necrotrophic stage, the strawberry cell contains elevated SA due to the increased expression of PAL1/2. SA-dependent resistance is partially activated after NPR1 binds SA. Enhanced NPR31 negatively regulates SA-related immunity. Meanwhile, SA-dependent resistance is antagonized by the disinhibition of MeJA-mediated defense and the activation of ethylene-related defense, which is beneficial for strawberry defending the pathogen with a necrotrophic life at 96 hpi. There exists monomeric NPR1 and NPR31 in the nucleus, which directly target TGA transcriptional factors to regulate the expression of PR genes. Members are in red characters for up-regulated and in blue for down-regulated.