Molecular Insights into the Role of Cysteine-Rich Peptides in Induced Resistance to Fusarium oxysporum Infection in Tomato Based on Transcriptome Profiling

Abstract

Cysteine-rich peptides (CRPs) play an important role in plant physiology. However, their role in resistance induced by biogenic elicitors remains poorly understood. Using whole-genome transcriptome sequencing and our CRP search algorithm, we analyzed the repertoire of CRPs in tomato Solanum lycopersicum L. in response to Fusarium oxysporum infection and elicitors from F. sambucinum. We revealed 106 putative CRP transcripts belonging to different families of antimicrobial peptides (AMPs), signaling peptides (RALFs), and peptides with non-defense functions (Major pollen allergen of Olea europaea (Ole e 1 and 6), Maternally Expressed Gene (MEG), Epidermal Patterning Factor (EPF)), as well as pathogenesis-related proteins of families 1 and 4 (PR-1 and 4). We discovered a novel type of 10-Cys-containing hevein-like AMPs named SlHev1, which was up-regulated both by infection and elicitors. Transcript profiling showed that F. oxysporum infection and F. sambucinum elicitors changed the expression levels of different overlapping sets of CRP genes, suggesting the diversification of functions in CRP families. We showed that non-specific lipid transfer proteins (nsLTPs) and snakins mostly contribute to the response of tomato plants to the infection and the elicitors. The involvement of CRPs with non-defense function in stress reactions was also demonstrated. The results obtained shed light on the mode of action of F. sambucinum elicitors and the role of CRP families in the immune response in tomato.

Keywords: F. sambucinum; Fusarium oxysporum f. sp. lycopersici; Solanum lycopersicum L.; antimicrobial peptides; cysteine-rich peptides; elicitors; high-throughput transcriptome sequencing (RNA-seq); plant immunity; signaling peptides.

Figure 1

Multiple sequence alignment of S. lycopersicum DEFL precursors and Arabidopsis thaliana AFP1 (NP_565119.1), Nicotiana alata NaD1 (AAN70999.1), and Solanum pennellii defensin D1-like (XP_015081705.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 2

Heatmaps of differentially expressed CRP genes. (A) Genes with expression levels above 50 CPM (Counts per Million Mapped Reads) at least in one transcriptome. (B) Genes with expression levels below 50 CPM in all transcriptomes. Cont, Inf-2, Inf-4, Ind, and IR designate control, infected at 2 or 4 dpi, induced and IR-displaying plants, respectively.

Figure 3

Sequence logo plots of aligned 8-cysteine motif sequences for each SlLTP type. The numbers on the x-axis represent the positions in the 8-cysteine motif. On the y-axis, the information content measured in bits is presented.

Figure 4

Family distribution of CRP genes, differentially expressed genes (DEGs), and genes up-regulated (IR/Inf↑) and down-regulated (IR/Inf↓) in IR-displaying plants compared to infected at 4 dpi.

Figure 5

Multiple sequence alignment of S. lycopersicum snakin precursors and Solanum tuberosum SN1 (Q948Z4.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 6

Multiple sequence alignment of S. lycopersicum thionin precursors and Arabidopsis thaliana thionin-2.1 (NP_565038.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 7

Multiple sequence alignment of hevein-like peptide precursors of S. lycopersicum SlHev1 and T. kiharae WAMP-1 (P85966.2), hevein of Hevea brasiliensis (1Q9B_A) and hevein-like peptides from Pharbitis nil Pn-AMP1 (P81591), E. ulmoides EAFP1 (P83596), E. europaeus Ee-CBP (Q7Y238), Fagopyrum esculentum Fa-AMP1 (P0DKH7), Amaranthus caudatus Ac-AMP2 (Q9S8Z7), A. retroflexus Ar-AMP (Q5I2B2), and Beta vulgaris IWF4 [34]. Cysteine residues are shaded black, and identical amino acids are shaded gray. The arrangement of disulfide bonds is shown above the alignment.

Figure 8

Multiple sequence alignment of S. lycopersicum hevein-like peptide precursor SlHev1 and homologous sequences of Solanum chilense (Sc), Capsicum baccatum (Cb), C. annuum (Ca), Nicotiana attenuata (Na), and C. chinense (Cc). Cysteine residues are shaded black, and identical amino acids are shaded gray. Sequence identity values are shown to the right of the alignment.

Figure 9

The 3D structure model of SlHev1 and the NMR solution structure of hevein (1HEV). Modeling was carried out using SWISS-MODEL [35]. Disulfide bonds are shown by thin yellow lines. The N-and C-termini are indicated by N and C, respectively. Cysteine residues are colored yellow and numerated according to their position in the polypeptide chain.

Figure 10

Multiple sequence alignment of S. lycopersicum knottin precursors and S. tuberosum metallocarboxypeptidase inhibitor precursor (mcpi) IIa (NP_001275048.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 11

Multiple sequence alignment of S. lycopersicum RALF precursors and RALF from Nicotiana tomentosiformis (XP_00962090.1), RALF 1 from A. thaliana (NP_171789.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 12

Multiple sequence alignment of S. lycopersicum MEG precursors and Zea mays MEG1 (XP_008652138.1), MEG2-like (XP_020397032.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 13

Multiple sequence alignment of S. lycopersicum Ole e 1 precursors and O. europaea Ole e 1-like (XP_022872526.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 14

Multiple sequence alignment of S. lycopersicum Ole e 6 precursors and C. baccatum Ole e 6 (PHT46953.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 15

Multiple sequence alignment of S. lycopersicum EPF precursors and A. thaliana EPF1 [51]. Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 16

Multiple sequence alignment of S. lycopersicum PR-1 precursors. Cysteine residues are shaded black, and identical amino acids are shaded gray. The mature CAPE1 peptide sequence is underlined.

Figure 17

Multiple sequence alignment of S. lycopersicum PR-4 precursor and Nicotiana tabacum PR-4A (P29062.1), Hordeum vulgare pathogenesis-related protein 4 (PR-4) (KAE8805200.1), and T. aestivum wheatwin-1 (O64392.1). Cysteine residues are shaded black, and identical amino acids are shaded gray.

Figure 18

qRT-PCR validation of expression levels for selected S. lycopersicum CRP genes. Relative expression values were normalized using the EF1-α gene as internal control and standardized relative to the control values. Analyses were accomplished in triplicate. Bars represent mean ± standard deviation (SD).

Figure 19

Venn diagram showing the number of CRP genes specifically up- or down-regulated compared to control in elicitor-treated (Ind), F. oxysporum-infected at 4 dpi (Inf), and IR-displaying S. lycopersicum plants as well as similarly expressed CRPs in all three transcriptomes. For up-regulated genes, expression fold change was ≥2, for down-regulated genes, it was ≤0.5.

Figure 20

Differentially expressed CRP genes in S. lycopersicum transcriptomes (as a percentage of the total number of expressed CRP genes). Up-regulated genes (expression fold change ≥2) are colored green; down-regulated genes (expression fold change ≤0.5) are colored pink; genes whose expression level did not change are shown in yellow. The designations below the figure are as follows: Inf/Cont, infected at 2 and 4 dpi versus control; Inf-4/Inf-2, infected at 4 dpi versus infected at 2 dpi; Ind/Cont, elicitor-treated versus control; IR/Cont, IR-expressing versus control; IR/Ind, IR-expressing versus elicitor-treated; IR/Inf-4, IR-expressing versus infected at 4 dpi; Inf-4/Ind, infected at 4 dpi versus elicitor-treated.

Figure 21

Venn diagram showing the number of CRP genes specifically up- or down-regulated in IR-displaying plants compared to elicitor-treated (Ind) and F. oxysporum-infected at 4 dpi (Inf) seedlings as well as similarly expressed genes in both transcriptomes. For up-regulated genes, expression fold change was ≥2, for down-regulated, it was ≤0.5.