Alfalfa snakin-1 prevents fungal colonization and probably coevolved with rhizobia

Abstract

Background: The production of antimicrobial peptides is a common defense strategy of living cells against a wide range of pathogens. Plant snakin peptides inhibit bacterial and fungal growth at extremely low concentrations. However, little is known of their molecular and ecological characteristics, including origin, evolutionary equivalence, specific functions and activity against beneficial microbes. The aim of this study was to identify and characterize snakin-1 from alfalfa (MsSN1).

Results: Phylogenetic analysis showed complete congruence between snakin-1 and plant trees. The antimicrobial activity of MsSN1 against bacterial and fungal pathogens of alfalfa was demonstrated in vitro and in vivo. Transgenic alfalfa overexpressing MsSN1 showed increased antimicrobial activity against virulent fungal strains. However, MsSN1 did not affect nitrogen-fixing bacterial strains only when these had an alfalfa origin.

Conclusions: The results reported here suggest that snakin peptides have important and ancestral roles in land plant innate immunity. Our data indicate a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria. The increased antimicrobial activity against virulent fungal strains without altering the nitrogen-fixing symbiosis observed in MsSN1-overexpressing alfalfa transgenic plants opens the way to the production of effective legume transgenic cultivars for biotic stress resistance.

Figure 1

Alignment of residues 26–91 of Ms SN1 from alfalfa with the corresponding regions of snakin/GASA proteins from potato ( St SN1), Arabidopsis (GASA7 and GASA8) and Peach (Peamaclein) revealing 12 cysteine residues in conserved positions within a conserved C-terminal region. These cysteine residues are shown in yellow.

Figure 2

Phylogenetic tree of plant SN1 protein sequences using the neighbor-joining method and root on midpoint. Bootstrap percentages are indicated at the branch points. The current classification of plants is found on the right. Tree topology obtained using NJ method, Minimum evolution and Maximum parsimony methods were identical. The MsSN1 peptide characterized in this work and other snakin/GASA peptides described in previous experimental studies are boxed.

Figure 3

Analysis of the in vitro antimicrobial activity of Ms SN1. Agrobacterium growth (A-B) and Phoma spore germination (1 × 10⁵ spores/ml) (C-F) inhibition assays using MsSN1-free (A, C and D) or MsSN1 extracts (B, E and F). Spore germination was examined immediately (C and E) and after 16 h (D and F) of incubation with the extracts. Bar = 50 μm.

Figure 4

Quantitative analysis of the expression of the MsSN1 gene in wild type alfalfa plants under microbe stress conditions. Real-time RT-PCR studies for MsSN1 expression in non-inoculated leaves and roots (NI) or in roots exposed to Agrobacterium tumefaciens LBA4404, Pseudomonas fluorescens Pf-5 and Sinorhizobium meliloti BL225C for 24 hs.

Figure 5

Characterization of transgenic alfalfa lines overexpressing MsSN1 . (A) Schematic representation of the T-DNA region of binary vector pART-35S::MsSN1 containing the MsSN1 gene under the CaMV 35S promoter. Relevant restriction enzymes used in plasmid construction and Southern Blot analysis are shown. RB: right border; CaMV 35S: promoter; Topo: region derived from pCR2.1-TOPO vector; UTRs: untranslated regions derived from the native MsSN1 gene; os-t: octopine synthase terminator; pnos-nptII-nos-t: kanamycin cassette (where, pnos and nos-t are nopaline synthase promoter and terminator, respectively); LB: left border. (B) Real-Time RT-PCR assays of alfalfa transgenic lines (S1-S3) and control untransformed plants (wt). All values are log means ± SEM (n = 3). Asterisks indicate a statistically significant difference (Tukey: ***p < 0.001).

Figure 6

Antifungal in vitro activity of MsSN1 overexpressing transgenic plants. (A) Diseased leaflets related to leaflets inoculated with Phoma (#diseased leaflet/#total leaflet). (B) Diseased leaflets related to leaflets inoculated with Colletotrichum (#diseased leaflet/#total leaflet). (C) Damage score: 1. Healthy leaflet, 2. Countable injuries, 3. Uncountable injuries, 4. Chlorosis, 5. completely damaged. (D) Representative Phoma assay. Wt: wild type. S1-S3: MsSN1 transgenic plants. All values are log means ± SEM (n = 10–30). Asterisks indicate a statistically significant difference (Turkey: *p < 0.5; **p < 0.01 ***p < 0.001). Leaflets were considered diseased when they showed symptoms 3, 4 or 5 in the damage score. Disease severity was estimated from scoring 30 to 60 detached leaflets from three individual plants.

Figure 7

In vivo characterization of MsSN1 overexpressing transgenic plants for resistance to Phoma medicaginis CT1. (A) Number of diseased leaflets related to total number of leaflet in 30-day-old inoculated plants. (B) Percentage of two-month-old inoculated plants with regrowth. (C) Percentage of vigor affected two-month-old inoculated plants. (D) Percentage of highly defoliated two-month-old inoculated plants (highly defoliated > 3 leaflets detached). (E) Representative photo comparing transgenic (S) with wild type (Wt) 30-day-old inoculated plants. (F) Detail of regrowth. (G) Detail of symptomatic regrowth. S: MsSN1-overexpressing plants. Wt: wild type. All values are means ± SEM (n =15-25). N.S: non-significant; *p < 0.05; **p < 0.01; ***p < 0.001, t-test.

Figure 8

Bacterial colonization of MsSN1 -overexpressing transgenic plants. Analysis of alfalfa-Sinorhizobium meliloti BL225C interactions: number of nodules in two-month-old alfalfa plants. (A) Photo of nodulated root. (B) Study of alfalfa colonization by Pseudomonas fluorescens Pf-5. (C) MsSN1-overexpressing plants. Wt: wild type. All values are means ± SEM (n =20). N.S: non-significant, ***p < 0.001, t-test.