Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant-pathogen interaction in transgenic Arabidopsis

Abstract

The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad-spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH-1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co-expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.

Figure 1

Nucleotide sequence of MtDef4.2 and the exon–intron maps of the MtDef4‐like gene family in Medicago truncatula. (a) Nucleotide and deduced amino acid sequence of MtDef4.2. The signal peptide is highlighted in turquoise and the mature peptide is highlighted in red. (b) Exon–intron arrangement of the MtDef4 gene family in M. truncatula. The size of each box is indicated in base pairs in parentheses.

Figure 2

Amino acid sequence alignment of the precursor MtDef4.1–4.7 defensins. Highly conserved cysteine residues involved in disulphide bridge formation are highlighted in yellow. Signal peptide is indicated in black italics.

Figure 3

Expression analysis of defensins MtDef4.1, MtDef4.2, MtDef4.5 and MtDef4.6 based on MtGEA (Medicago truncatula Gene Expression Atlas). Expression in various tissues of M. truncatula (a) and at different stages of seed development and in the seed coat (b) is shown. dap, days after pollination.

Figure 4

Neighbour‐joining phylogram obtained using the amino acid sequences of MtDef4.1–4.7 and their homologues from other plants already published (reviewed in 2008; Lay and Anderson, 2005). The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analysed using the DNAstar Lasergene program. The boxes indicate the position of Medicago truncatula defensins.

Figure 5

Expression vectors used for the subcellular targeting of MtDef.4.2 in transgenic Arabidopsis. Chimeric MtDef4.2 gene constructs were driven by the constitutive figwort mosaic virus (FMV) 35S promoter and terminated by nos‐3′. The MtDef4.2‐Ec construct directs the protein to the extracellular space. The MtDef4.2‐Vc construct for vacuolar targeting and the MtDef4.2‐ER construct for endoplasmic reticulum (ER) retention contain the barley lectin carboxy‐terminal propeptide (CTPP) signal sequence and the KDEL sequence, respectively, fused in frame to the carboxy‐terminal cysteine residue of mature defensin. nos, nopaline synthase.

Figure 6

Expression analysis of MtDef4.2 in 5‐week‐old seedlings of transgenic Arabidopsis lines using quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). Normalization was performed by comparing the expression of A. thaliana housekeeping gene Ubi10 with respect to the wild‐type (WT). Error bars represent the standard error (SE) of three replicates.

Figure 7

Immunofluorescence labelling of MtDef4.2 in the seed of transgenic Arabidopsis lines. (a, b, inset) Line Ec4‐3 targeting MtDef4.2 to the cell wall (arrowheads). Note the intracellular signal (arrows) showing protein being transported through the cell endomembrane system. (c, d, inset) Line Vc9‐1 with vacuolar targeting. The signal is weak in the vacuoles, but can be seen in punctate structures (arrows) that could be small vacuoles or endomembrane system components transporting MtDef4.2 to the vacuoles. (e, f, inset) Line ER16‐4 with MtDef4.2 retained in the endoplasmic reticulum (ER); arrows show the localization equivalent to that of ER‐targeted green fluorescent protein (GFP) in cotyledon cells shown in (g). V, vacuoles; N, nucleus; scale bar for all panels, 5 μm.

Figure 8

Transgenic Arabidopsis lines challenged with Hyaloperonospora arabidopsidis Noco2 at 7 days post‐inoculation (dpi). (a) Representative leaves of wild‐type (WT) and transgenic line Ec4‐3. White arrows indicate fungal hyphae, black arrows conidiophores, red arrows trailing necrosis (indicative of failed infection attempt) and red asterisk conidiospores. Scale bar for both panels, 200 μm. (c) Analysis of variance (anova) of spore count data followed by a Tukey analysis. Three transgenic Arabidopsis lines belonging to each construct were combined for this analysis. Different letters indicate statistically significant differences (P < 0.05).

Figure 9

Modified scale of 0–7 used to score Fusarium graminearum‐inoculated siliques: 0, normal; 1, bleaching of surface; 2, aerial mycelium growing on surface; 3, drying of most of the silique; 4, aerial mycelium growth accompanied by drying; 5, bleaching advancing to pedicle of silique; 6, mycelial growth on pedicle, occasional splitting of silique; 7, loss of silique by disease travelling down the stem and main stem constriction (not shown).

Figure 10

Relative percentages of wild‐type (WT) and transgenic siliques with different disease scores inoculated with Fusarium graminearum in three different experiments.

Figure 11

Trypan blue‐stained siliques inoculated with Fusarium graminearum and fungal biomass quantification at 8 days post‐inoculation (dpi). (a) Representative siliques of wild‐type (WT) and transgenic lines Ec4‐3, Vc25‐8 and ER16‐4. White and yellow arrows point to fungal hyphae and spores, respectively. Scale bar for all panels, 50 μm. (e) Fungal biomass was calculated in the inoculated siliques using quantitative polymerase chain reaction (qPCR).