Alfalfa snakin-1 prevents fungal colonization and probably coevolved with rhizobia
© García et al.; licensee BioMed Central Ltd. 2014
Received: 20 June 2014
Accepted: 11 September 2014
Published: 17 September 2014
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).
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.
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.
Alfalfa (Medicago sativa L.), known as the “Queen of Forages”, is a perennial legume. This species is native to Asia, and is considered one of the first known crops with a cultivation history of at least 3500 years. Due to its strong vitality, high nutritional quality, high yields, high adaptability and multiple uses, alfalfa is the main forage crop produced in temperate regions of the planet. Elite alfalfa cultivars must not only have high forage yields but also maintain their productivity and stands over several years to provide substantial economic benefits. Regarding this complex topic, improved fungal disease resistance has been specifically identified as the critical trait in alfalfa persistence . In this context, it is proposed that the use of snakin-1 peptide (SN1), a powerful but poorly studied antimicrobial compound, to improve alfalfa tolerance to virulent fungal pathogens should be explored.
Antimicrobial peptides are present in virtually all organisms and are an ancient and critical component of innate immunity. SN1, the first member of the snakin family to be characterized, was isolated from a crude cell wall preparation of potato (Solanum tuberosum) tubers (StSN1) . This cysteine-rich peptide from potato was found to be active against bacterial and fungal pathogens at extremely low concentrations (EC50 < 10 μM) . The expression pattern of the StSN1 gene suggests that plant SN1 could be a component of constitutive defense barriers, especially those of storage and reproductive plant organs . A second snakin peptide (StSN2), which has a high amino acid identity (30%) to StSN1, was also isolated from a crude cell wall preparation of potato tubers. Consistent with this high amino acid identity, StSN2 is also active at very low concentrations against a wide range of pathogens . In contrast to StSN1, the expression of StSN2 is locally induced by wounding and pathogen infection, suggesting a critical role of snakin-2 in both constitutive and inducible defense barriers of plants. These strong antimicrobial activities of snakin peptides have been verified using bacterial and eukaryotic heterologous expression systems -. Based on the presence of the Gibberellic Acid Stimulated Arabidopsis (GASA) domain and the absence of bioinformatic (e.g. RGD residues) and functional (e.g. toxic activity) data supporting its relationship with cysteine-rich peptides from snakes venoms, StSN1 and StSN2 have been recently renamed as GSL1 and GSL2, respectively ,.
Overexpression of SN1 in potato and wheat (Triticum aestivum), SN2 in potato and tomato (Solanum lycopersicum), and snakin-defensin hybrid protein in tobacco (Nicotiana tabacum) and potato restricts pathogen invasiveness and enhances tolerance to bacterial and fungal diseases, without altering the agronomic phenotype of these crops ,-. Furthermore, disease sensitivity is enhanced by silencing SN2 in wild tobacco (Nicotiana benthamiana), supporting the central role of snakin peptides in plant defense . In addition, it has been recently shown that StSN1 is located in the plant cell wall , confirming that snakin peptides are components of the physical barrier and the first line of defense used by plant cells to prevent bacterial and fungal entry . Furthermore, in concordance with the functional classification of snakin peptides as members of the GASA protein family ,,, StSN1 silencing affects cell division, primary metabolism, and cell wall composition . Snakin peptides could have additional functions in plant growth and development beyond their demonstrated function in biotic stress response. In spite of their hypothetical functional similarity, there is little to no phylogenetic reports on the relationship between StSN1 and GASA-related proteins.
The aim of this study was to identify and characterize snakin-1 (MsSN1) gene of alfalfa. The phylogenetic and functional analyses showed here, propose that MsSN1 is an ancestral plant gene involved in biotic stress resistance, suggesting a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria.
Bacterial and fungal strains
The bacterial strains (all Gram-negative bacteria) used in this study were: Pseudomonas fluorescens Pf-5 , Sinorhizobium meliloti BL225C , Sinorhizobium meliloti SM11 , Sinorhizobium medicae WSM419 , Sinorhizobium fredii USDA 257 , Rhizobium sp. Or 191 , Rhizobium etli CFN 42 , Mesorhizobium loti MAFF303099 , Bradyrhizobium japonicum USDA110  and Agrobacterium tumefaciens LBA4404 . The fungal strains used in this work were: Phoma medicaginis strain CT1 and Colletotrichum trifolii strain CT2, isolated from INTA alfalfa cultivars and kindly provided by Dr. Ricardo Comerio (Instituto de Microbiología y Zoología Agrícola, Instituto Nacional de Tecnologia Agropecuaria, Argentina).
Two fungal pathogens, Phoma medicaginis strain CT1 and Colletotrichum trifolii strain CT2, were grown on PDA (Cat. # B0216605, Britania) plates at room temperature for approximately 7 days before the start of the bioassay. For spore collection, the plates were flooded with sterile distilled water and scraped with a wire loop. Spore concentration was adjusted to 1 × 106 spores/ml for C. trifolii CT2 and to 1 × 105 spores/ml for P. medicaginis CT1 with sterile distilled water. The fungal strains were maintained through sequential passages in plants.
The Medicago sativa plants used were the regenerative clone C2-3, kindly provided by Drs. B. McKersie and S. Bowley (Plant Biotechnology Division, Department of Plant Agriculture, University of Guelph, Canada), and the regenerative clone 432-19-17, previously isolated in our laboratory.
Bacterial and plant RNA Isolation and cDNA synthesis
Total bacterial (E. coli) and plant tissues (roots, steam, leaflets) RNA was extracted by using an RNeasy Mini Kit (Cat. # 74106, Qiagen) following the manufactures’ instructions. Samples of 2 μg total RNA isolated from bacterial cells or plant tissues were reverse-transcribed in a 25 μl reaction using MMLV-RT (Cat. # M1701, Promega). For PCR amplification in bacteria and plants, 1 μl of RT reaction was used. The PCR reactions were carried out in 25 μl with 0.5 μM of each primer , using Taq polymerase (Cat#. 11615010, Invitrogen) following the manufactures’ instructions.
MsSN1 plasmid construction
The MsSN1 cDNA (GenBank accession number JQ809686) was isolated using primers p1 FW and p2 RV (Additional file 1) designed against the 5′ and 3′ untranslated regions (UTR) of the putative SN1 gene (GenBank accession number XM_003589066, MTR_1g018640) from Medicago truncatula. Full-length cDNA was amplified by PCR and this fragment was cloned into a pCR2.1TOPO vector (Cat. # K4500-01, Invitrogen). The pTOPO-MsSN1 plasmid was digested with Not I (Cat. # R6431, Promega), treated with Klenow, and religated to destroy the polylinker Not I site. The resulting plasmid was named pTOPO-NotI-MsSN1. The sequencing reactions of pTOPO-NotI-MsSN1 were performed at INTA-Argentina (www.inta.gov.ar). The cDNA sequence was named MsSN1 (Medicago sativa snakin-1). To produce recombinant bacteria expressing MsSN1, plasmid pSJ33-MsSN1 carrying MsSN1 was constructed by subcloning the 0.5-kb EcoRI (Cat. # R6011, Promega) fragment from pCR2.1TOPO-MsSN1 into pSJ33 , and afterward introduced in Escherichia coli for heterologous expression of MsSN1. To produce transgenic alfalfa lines overexpressing MsSN1, pCR2.1TOPO-NotI-MsSN1 was digested with KpnI (Cat. # R6341, Promega) and XbaI (Cat. # R6181, Promega), and the MsSN1 restriction fragment was cloned into pKANNIBAL vector (GenBank accession number AJ311873). The resulting plasmid was digested with NotI, and the 35SMsSN1 restriction fragment was cloned into pART27 binary vector . The resulting recombinant binary vector containing the MsSN1 cDNA with its signal peptide under the CaMV 35S promoter was named pART-35S::MsSN1.
Bioinformatic analysis of MsSN1
MsSN1 sequence of Medicago sativa (AFE82743), a peptide composed of 91 amino acids, was used as query to search against all available complete eukaryotic genome databases in NCBI with protein annotation in GenBank. The cut-off to obtain candidate orthologs was 20% of amino acid identity (Additional file 2). Sequences were searched by using BLASTP tools in NCBI and PLAZA databases (http://www.ncbi.nlm.nih.gov/blast; http://bioinformatics.psb.ugent.be/plaza). Protein identity calculations were performed using MatGAT v2.02 . Evolutionary analysis was conducted by using MEGA version 5.0 . Protein sequences were aligned using the ClustalW program. Phylogenetic trees were constructed using the neighbor-joining method with genetic distances computed using the pairwise deletion model and bootstrap analysis of 500 values and root on midpoint (i.e. midpoint of the longest pathway between two clusters of sequences). In silico analysis of conserved motifs in putative snakin/GASA proteins, Pfam domains and signal peptides were predicted by using Pfam and Signal-3 L with default parameters, respectively ,.
DNA extraction and sequence analysis of fungal strains
Fungal DNA was extracted as previously described by Moller  with modifications suggested by Dr. E.W. Boehm (http://www.eboehm.com/). Briefly, 100 mg of fungal mycelia was scraped from 10-day-old PDA cultures, ground in a 1.5 ml tube with micropestle by adding 500 μl of Lysis Buffer (100 mM Tris pH 8, 10 mM EDTA, 2% SDS, 1% β-Mercaptoethanol, 100 μg/ml proteinase K). Lysate was incubated at 60°C for 60 min. Subsequently, 5 M NaCl was added to a final concentration of 1.4 M and mixed before adding 0.1 vol of CTAB 10% (w/v). The mixture was incubated at 65°C for 10 min. DNA was extracted by adding an equal volume of chloroform:isoamyl alcohol (24:1), incubating for 30 min at 0°C and centrifuging at 14,000 × g for 10 min at 4°C. The aqueous phase was mixed with 0.5 vol of 5 M ammonium acetate, incubated at 0°C for 60 min and centrifuged at 14,000 × g for 1 min. DNA was precipitated from the supernatant by adding 0.55 vol of isopropanol, centrifuged at 14,000 × g for 10 min and washed twice with 70% ethanol. DNA pellet was air-dried and resuspended in 50 μl of TE (10 mM Tris pH 8.0; 1 mM EDTA pH 8.0). The internal transcribed spacer regions 1 & 2 and 5.8S nrDNA (ITS) and partial sequences of TUB genes were amplified and sequenced by Macrogen Inc. (Korea) service (http://www.macrogen.com/) using the primer pairs ITS-1FW and ITS-4RV and Btub2FW and Btub4RV, respectively (Additional file 1). The nucleotide sequences obtained here were deposited in the EMBL Nucleotide Sequence Database, accession numbers: KF846005 for C. trifolii TUB, KF846006 for P. medicaginis TUB, KF846009 for C. trifolii ITS and KF846010 for P. medicaginis ITS.
In vitro antimicrobial activity assays
Escherichia coli recombinant strains containing the pSJ33 empty vector or pSJ33-MsSN1 were grown overnight at 30°C with shaking (250 rpm) in LB medium supplemented with 1 mM isopropyl-β-d-thiogalactopyranoside in 250-ml Erlenmeyer flasks containing 50 ml of medium. Samples of 25 ml of culture broth (O.D: 0.8) were centrifuged at 5,000 × g at 4°C for 10 min. The pellets were washed three times with physiological solution (0.9% NaCl). Crude cell lysates were achieved by four consecutive cycles of freezing in liquid nitrogen followed by thawing at 37°C. After centrifugation at 14,000 × g at 4°C for 20 min, the supernatant was resuspended in 400 μl of physiological solution. For the analysis of MsSN1 expression in E. coli, total bacterial RNA was extracted by using the RNeasy Mini kit and treated with DNaseI (Cat. # M6101, Promega). cDNA was obtained using random hexamers (Cat. # B070-40, Promega) and AMV Reverse Transcriptase (Cat. # M9004, Promega). For PCR amplification, 1 μl of RT reaction was used. The PCR reactions were carried out in 25 μl with 0.5 μM of each primer according to Setten . PCR conditions comprised: 1 cycle at 94°C for 3 min, 34 cycles of 94°C for 45 s, 56°C for 1 min and 72°C for 1 min. The expression of MsSN1 was analyzed using primers p3 FW and p4 RV which amplify the complete open reading frame (Additional file 1).
The disk inhibition assays were evaluated as described by Ayub , with very slight modifications. Cultures were performed in 125-ml Erlenmeyer flasks containing 25 ml of TY medium  or LB medium for rhizobia or Pseudomonas and Agrobacterium, respectively. Bacteria were incubated overnight at 28°C with shaking (250 rpm). Sterile Whatman No. 1 filter disks (5 mm) impregnated with 5 μl of MsSN1-free (E. coli pSJ33) or MsSN1 (E. coli pSJ33-MsSN1) extracts were placed on top of bacteria-seeded plates. Zones of inhibition were measured after incubation at 28°C for 24 h. The antifungal activities of the MsSN1 extract were determined according to Kovalskaya  by counting germinating and non-germinating fungal spores. The fungal spores of Phoma medicaginis var. medicaginis CBS 316.90 from CBS-KNAW Fungal Biodiversity Center (www.cbs.knaw.nl/Collections) were prepared in PDB. For the inhibition assays, spore suspensions of 1×105 spores/ml were used. Each antifungal assay was performed in triplicate.
The recombinant binary vector pART-35S::MsSN1 was introduced into Agrobacterium tumefaciens LBA 4404 by electroporation, by using the procedure described by Shen & Forde . Petioles of alfalfa clone C2-3 were transformed with pART-35S::MsSN1 via A. tumefaciens and cultured in vitro as described by D’Halluin , with slight modifications (Additional file 3). Petiole tissues were decontaminated by immersing in 70% ethanol for 1 min and then in 2% sodium hypochlorite for 20 min. The petioles were washed 3 times in sterile distilled water. Explants previously injured with a scalpel were inoculated with a bacterial culture for 2 minutes (OD600 mm = 0.5-0.8), and then dried in Whatman filter paper and transferred onto a solid co-cultivation SHK medium [3% sucrose, 0.435% KSO4, 2 mg/l 2.4-D, 0.2 mg/l kinetine, 6.5 g/l agar, 20% SHK stock solution (w/v) pH:5.8 (300 mg/l NH4H2PO4, 2.5% KNO3, 200 mg/l CaCl2.2H2O, 400 mg/l MgSO4.7H2O, 4.3% K2SO4, 1 mg/l KI, 5 mg/l H3BO3, 10 mg/l MnSO4.H2O, 1 mg/l ZnSO4.H2O, 1 mg/l Na2MoO4.2H2O, 1 mg/l CuSO4.5H2O, 0.1 mg/l CoCl2.6H2O, 26.29 mg/l NaFeEDTA.H2O, 288 mg/l proline, 53 mg/l thioproline, 200 mg/l myo-inositol, 5 mg/l nicotinic acid, 0.5 mg/l pyridoxine, 5 mg/l thiamine)] containing 100 μM acetosyringone for 2 days at 25°C in the dark. The explants were then washed with 0.5 g/l cefotaxime supplemented with distilled water and transferred to selection/induction medium SHK containing 25 mg/l kanamycin and 400 mg/l cefotaxime with routine transfers to fresh medium every 2 weeks, at 25°C and 16 h light (100 μmoles m−2 s−1). Somatic embryos were obtained three months later and then transferred to MS rooting medium, composed of Murashige and Skoog Basal Medium (Cat. # M5519, Sigma) diluted 1:2 with water. Transgenic plants were obtained about 6 months after callus induction. The regenerated plantlets were transferred to the greenhouse once they were well rooted. All plants were grown in a greenhouse at 25-20°C day/night temperature and watered daily. Alfalfa transgenic events (named S1, S2 and S3) were propagated by crossing with clone 432-19-17 and by cuttings to increase the number of plants available for biochemical, physiological, and genetic analysis.
Identification of transgenic plants
DNA was isolated from leaf tissue with the DNeasy plant mini kit (Cat. # 69104, Qiagen). Transgenic plants were first identified by PCR with primers p5 FW and p6 RV (Additional file 1), designed against the 35S promoter and SN1 regions of recombinant binary vector. The expression of the transgene was corroborated by RT-PCR, using primers p7 FW and p4 RV, designed against the pCR2-1-TOPO-MCS-derived 5′ UTR region and the 5′ UTR of the MsSN1 gene (Additional file 1). All PCR reactions were performed with Taq (Cat. # 11615010, Invitrogen).
For Southern hybridization analysis, genomic DNA was isolated from leaf tissues of greenhouse-grown plants using the DNeasy Plant Maxi kit (Cat. # 68161, Qiagen) following the manufacture′s indications. DNA was digested with the KpnI restriction enzyme, which cleaves the construct only once. Then, 20 μg of DNA from each sample was digested overnight and blotted after separation on 1% (w/v) agarose gel 1× TAE. The DNA fragments in gels were transferred to a positively charged Nylon membrane (Cat. # 11209272001, Roche). Nylon membranes were crosslinked and then used for hybridization with a DIG-labeled probe. Prehybridization and hybridization was carried out according to the manufacturer’s instructions. The hybridization probe MsSN1digoxigenin-labeled DNA was generated by PCR by using the PCR DIG probe synthesis kit (Cat. # 11573152910, Roche), using primers p8 FW and p9 RV and then used as a probe (Additional file 1). PCR amplification was performed under standard conditions (25 μl volume using 0.8 μM of each primer, 1X PCR buffer, 0.2 mM each dNTP, 2 mM MgCl2 and 20 ng of template) with a program of 34 cycles of 94°C for 1 min, 50°C for 30 s and 72°C for 2.5 min and a final cycle of 72°C for 10 min.
Real-time quantitative RT-PCR (RT-qPCR)
For RT-qPCR, PCR amplification was performed with 5 μl of RT (1:5 diluted) per reaction, by using 1 U iQ SYBR green Supermix (Cat. # 170–8880, Bio-Rad) and 0.2 mM primers, with the iCycler iQ system. Primers for the real-time qPCR were p10 FW and p11 RV (Additional file 1). qPCR conditions comprised: 1 cycle at 94°C for 5 min, 34 cycles of 94°C for 45 s, 59.1°C for 30 s, and 72°C for 30 s. At each cycle, accumulation of PCR products was detected. The amplification fragment was sequenced and found to be identical (100% nucleotide identity) to the MsSN1 gene. The expression level of MsSN1 was normalized using aspartate aminotransferase (ATT) (AAB46610) as a housekeeping gene, using primers p12 FW and p13 RV (Additional file 1). The efficiency of primer binding was determined by linear regression by plotting the cycle threshold value versus the log of the cDNA dilution . RT-qPCR experiments were performed two or three times with independent RNA samples (biological replicates). For each biological replicate the qPCR reactions were carried out in duplicate.
Bioassays. In vitro challenges
Assays were performed in healthy 15 day-old non-fumigated leaflets. Leaflets were decontaminated washing them in flasks with sterile water with Tween-20 (0.01%) for 10 min. After three washes with sterile water, the leaflets were transferred to a 5 % hypochlorite solution for 5 min. Finally, leaflets were washed three times with sterile water and transferred to agar-water petri dishes. Plates were maintained in growth chambers programmed for 16 h light at 23°C and 8 h dark at 20°C. Leaflets were inoculated by putting 5 μl of P. medicaginis CT1 or C. trifolii CT2 spore solution per leaflet. Infection evolution was observed and documented by photos and the following damage score was generated to quantify injuries: 1. Healthy leaflet, 2. Countable injuries, 3. Uncountable injuries, 4. Chlorosis, 5. Completely damaged.
Bioassays. In vivo challenges
Alfalfa transgenic seeds were obtained by crossing transgenic plants S1, S2 and S3 with 432-19-17 genotype plants. Transgenic and wild type seeds were treated with sulfuric acid for 10 minutes, washed three times with sterile water and placed in petri dishes with 1% agar water at 16 h of light (100 μmoles/m2s) and 25°C. Germinated plantlets were transferred to MS 0.5X flasks and incubated at 25°C with 16 h photoperiod for 1 month, after which plants were transferred to 1:1 vermiculite:perlite and maintained in magenta vessels (SIGMA) to conserve the humidity. Two-month-old plants were inoculated with P. medicaginis CT1 by spraying spore suspension to all aerial tissues. Percentage of diseased leaflet was analyzed 30-day post-inoculation and the number of plants with regrowth and the percentage of highly defoliated plants was counted 60-days post-inoculation. As an additional severity parameter of plant disease, the percentage of vigor affected plants (i.e. with visual signs of turgidity loss compared with non-inoculated plants) was estimated. All experiments were carried out with at least 10 independent plants per treatment and with at least 5 non inoculated wild type plants used as control.
Evaluation of bacterial root colonization
For the symbiosis assay, wild type and transgenic alfalfa seedlings were grown in 100% vermiculite and daily irrigated with the minimal medium called “INTA13 without nitrogen” . 10-days-old plants were inoculated with an early stationary-phase culture of rhizobial suspension (Sinorhizobium meliloti BL225C). After a month, the plants were harvested and the number of pink nodules was analyzed. Non-inoculated plants were used as controls. Three replicates were analyzed for each treatment. The P. fluorescens Pf-5 colonization assay was evaluated according to Sanchez , with two slight modifications: alfalfa was grown in INTA13 supplemented with Ca (NO3)2 and 0.5 NE2 medium plus sodium octanoate (0.25% w/v) was used to estimate the colony forming units .
Biological measurements were repeated at least three times with at least 10 different plants. Significant differences between treatments were calculated using Student’s t-test. qPCR experiments were independently performed three times, with comparable results. The three PCR reactions were carried out in duplicate. Significant differences were calculated using ANOVA followed by Tukey test.
Results and discussion
Identification and evolutionary analysis of MsSN1
In order to support the vertical origin of higher plant SN1, the SN1 gene is present in the primitive vascular plant Selaginella moellendorffii (Figure 2). In addition, an exhaustive phylogenetic analysis of proteins within genome databases failed to find orthologous genes of SN1 in the ancestral moss Physcomitrella patens, and in green algae species belonging to the phylum Chorophyta such as Ostreococcus lucimarinus, Micromonas sp. RCC299, Volvox carteri and Chlamydomonas reinhardtii. In agreement with this finding, proteins containing the snakin/GASA domain are widely distributed in land plants but completely absent in moss and green algae (data not shown), suggesting that SN1 is an ancestral gene that appeared in the vascular lineage after the vascular/bryophyte separation. This is not surprising considering that the pioneering ancestors of land plants that dominated terrestrial environments had to confront dramatic stresses, including the infection produced by an extreme diversity of microbial soil communities, selecting a number of genetic innovations . Therefore, the emergence of SN1 could be an adaptation of ancestral plants to land.
Analysis of the in vitro antimicrobial activity of MsSN1
Analysis of MsSN1 expression
Molecular characterization of transgenic alfalfa plants overexpressing MsSN1
Similar to transgenic lines from potato, wheat and tomato ,,, overexpression of MsSN1 did not appear to affect negatively the general phenotype in transgenic alfalfa plants, e.g. there was no reduction in the plant vigor in transgenic plants compared to wild type plants (Additional file 8). Therefore, transgenic alfalfa plants appear to be a suitable platform to study the role and biotechnological potential of MsSN1. Unfortunately, no MsSN1 reduced expression or silenced alfalfa plants were obtained (data not shown). Similar results were reported by Meiyalaghan  where the authors found no regenerant plants using antisense vectors expressing GSL1 (StSN1) and GSL2 (SN2) under their endogenous promoters regulation. These results probably indicate that MsSN1 silencing had detrimental effects on transformation or regeneration efficiency. In concordance with this observation, SN1 silencing drastically affects the cell division and primary metabolism of potato plants .
Molecular characterization of two highly virulent alfalfa fungal strains
Anthracnose and spring leaf spot, caused by the fungal pathogens Colletotrichum trifolii and Phoma medicaginis, respectively, are the most destructive infections of alfalfa worldwide. In the analysis of alfalfa resistance to these fungal diseases, there is usually a tradeoff between virulence and characterization of these pathogenic strains. Generally, long-term successive passages of archetype strains in vitro attenuate the virulence of these pathogens in vivo, while pathogens freshly isolated present a high virulence phenotype but their taxonomic position is commonly speculated based on disease symptoms or microscopy observations. In order to bypass this constraint, we used two highly virulent alfalfa fungal strains from alfalfa breeding programs, maintained through sequential plant infections and characterized through amplification, sequencing and phylogenetic analyses of conserved genes and intergenic regions. The evolutionary analyses of ITS region and TUB gene showed that these isolates belong to Colletotrichum and Phoma (Additional file 9). In fact, these DNA fragments from virulent strains (KF846005, KF846006, KF846009 and KF846010) share 100% nucleotide identity with the ITS region and TUB genes from C. trifolii CBS 158.83 (KF178478 and KF178599) and P. medicaginis CBS 316.90 (GU237828 and GU237630), respectively. In accordance with these data, these isolates were named Colletotrichum trifolii strain CT2 and Phoma medicaginis strain CT1. Unlike the well-characterized fungal strains C. trifolii CBS 158.83 and P. medicaginis CBS 316.90, strains C. trifolii CT2 and P. medicaginis CT1 are highly virulent on alfalfa and infect a wide range of commercial cultivars as well as the alfalfa clones used in this work, C2-3 and 432-19-17. This confirms that the form of conservation of fungal pathogens is essential to maintain their alfalfa virulence, and therefore, to study the genetic basis of resistance to fungi in alfalfa.
Antifungal in vitro activity of MsSN1-overexpressing transgenic plants
Antifungal in vivo activity of MsSN1-overexpressing transgenic plants
The three transgenic alfalfa lines (S1-S3) were chosen for progeny analysis of their potentially enhanced biotic resistance. The clone 432-19-17 was used as a pollen donor in a sexual cross with the transgenic lines to avoid the common inbreeding depression of progeny from self-crossed alfalfa plants. The presence and expression of the transgene in leaves of the progeny plants were assayed by PCR and RT-qPCR analyses, respectively (data not shown). In vivo assays for antifungal activity against P. medicaginis CT1 were carried out spraying a spore solution to transgenic and wild type plants. Plant disease severity was estimated by evaluating agronomic parameters for two months. Wild type plants manifested all the symptoms of the disease ,, including dark brown lesions on the stems, dark brown lesions on the leaflets that begin with specific dark color injuries, wilting leaves, chlorosis, and defoliation. Transgenic lines presented scarce symptoms and usually looked like control plants (data not shown).
Coevolution evidences of MsSN1 and rhizobia
Antimicrobial activity of alfalfa snakin-1 extract on rhizobial strains
Source of the strain
Zone of inhibition (mm)
Bradyrhizobium japonicum USDA110
1.38 ± 0.13
Mesorhizobium loti MAFF303099
1.50 + 0.30
Rhizobium sp. Or 191
Rhizobium etli CFN 42
1.18 + 0.09
Sinorhizobium meliloti BL225C
Sinorhizobium meliloti SM11
Sinorhizobium medicae WSM419
1.26 ± 0.06
Sinorhizobium fredii USDA 257
1.13 ± 0.13
There is a fine line between bacterial symbiosis and chronic infection. While one is beneficial the other is detrimental. Recent findings suggest that both share mechanisms for sidestepping host defenses . Legumes evolved rapidly and shortly after their origin, and nodulation most likely evolved several times during their divergence . These plants have symbiotic nitrogen-fixing bacteria living in root nodule compartments that also contain antimicrobial compounds. To avoid infection with phytopathogenic bacteria, nitrogen-fixing rhizobial bacteria and leguminous plants have developed complex signal exchange mechanisms that allow a specific bacterial strain to induce its specific host plant species to form invasion structures through which the bacteria can enter the plant root . The evidence of co-evolution presented here suggested that MsSN1 peptide could be part of the battery of compounds involved in discriminating the symbionts from other microbes, so as to allow the beneficial infection and inhibit colonization of potentially pathogenic microbes.
Previous studies and the results reported here show that snakin peptides have important and ancestral roles in land plant innate immunity. Interestingly, MsSN1-overexpressing alfalfa transgenic plants show increased antimicrobial activity against virulent fungal strains without altering the nitrogen-fixing symbiosis, opening the way to the production of effective alfalfa transgenic cultivars for biotic stress resistance. In this work, data also suggest a coevolutionary process, in which alfalfa exerts a selection pressure for resistance to MsSN1 on rhizobial bacteria.
In memory of Ing. Agr. Raúl Ríos.
We thank Carmen Soria, Juan Riquelme and Guillermo Piparola (IGEAF-INTA) for their technical support.
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica (grant PICT 2011–1325). NDA; CAB; JPM and GCS are researchers of the National Scientific and Technical Research Council (CONICET).
- Volenec JJ, Cunningham SM, Haagenson DM, Berg WK, Joern BC, Wiersma DW: Physiological genetics of alfalfa improvement: past failures, future prospects. Field Crops Res. 2002, 75: 97-110. 10.1016/S0378-4290(02)00020-5.View ArticleGoogle Scholar
- Lopez-Solanilla E, Garcia-Olmedo F, Rodriguez-Palenzuela P: Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis. Plant Cell. 1998, 10 (6): 917-924.PubMed CentralPubMedGoogle Scholar
- Segura A, Moreno M, Madueno F, Molina A, Garcia-Olmedo F: Snakin-1, a peptide from potato that is active against plant pathogens. Mol Plant Microbe Interact. 1999, 12 (1): 16-23. 10.1094/MPMI.19126.96.36.199.View ArticlePubMedGoogle Scholar
- Berrocal-Lobo M, Segura A, Moreno M, Lopez G, Garcia-Olmedo F, Molina A: Snakin-2, an antimicrobial peptide from potato whose gene is locally induced by wounding and responds to pathogen infection. Plant Physiol. 2002, 128 (3): 951-961. 10.1104/pp.010685.PubMed CentralView ArticlePubMedGoogle Scholar
- Kovalskaya N, Hammond RW: Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins. Protein Expr Purif. 2009, 63 (1): 12-17. 10.1016/j.pep.2008.08.013.View ArticlePubMedGoogle Scholar
- Rong W, Qi L, Wang J, Du L, Xu H, Wang A, Zhang Z: Expression of a potato antimicrobial peptide SN1 increases resistance to take-all pathogen Gaeumannomyces graminis var. tritici in transgenic wheat. Funct Integr Genomics. 2013, 13 (3): 403-409. 10.1007/s10142-013-0332-5.View ArticlePubMedGoogle Scholar
- Guzman-Rodriguez JJ, Ibarra-Laclette E, Herrera-Estrella L, Ochoa-Zarzosa A, Suarez-Rodriguez LM, Rodriguez-Zapata LC, Salgado-Garciglia R, Jimenez-Moraila B, Lopez-Meza JE, Lopez-Gomez R: Analysis of expressed sequence tags (ESTs) from avocado seed (Persea americana var. drymifolia) reveals abundant expression of the gene encoding the antimicrobial peptide snakin. Plant Physiol Biochem. 2013, 70: 318-324. 10.1016/j.plaphy.2013.05.045.View ArticlePubMedGoogle Scholar
- Meiyalaghan S, Thomson SJ, Fiers MW, Barrell PJ, Latimer JM, Mohan S, Jones EE, Conner AJ, Jacobs JM: Structure and expression of GSL1 and GSL2 genes encoding gibberellin stimulated-like proteins in diploid and highly heterozygous tetraploid potato reveals their highly conserved and essential status. BMC Genomics 2014, 15:2.,PubMed CentralView ArticlePubMedGoogle Scholar
- Mohan S, Meiyalaghan S, Latimer JM, Gatehouse ML, Monaghan KS, Vanga BR, Pitman AR, Jones EE, Conner AJ, Jacobs JM: GSL2 over-expression confers resistance to Pectobacterium atrosepticum in potato. Theor Appl Genet. 2014, 127 (3): 677-689. 10.1007/s00122-013-2250-2.View ArticlePubMedGoogle Scholar
- Almasia NI, Bazzini AA, Hopp HE, Vazquez-Rovere C: Overexpression of snakin-1 gene enhances resistance to Rhizoctonia solani and Erwinia carotovora in transgenic potato plants. Mol Plant Pathol. 2008, 9 (3): 329-338. 10.1111/j.1364-3703.2008.00469.x.View ArticlePubMedGoogle Scholar
- Balaji V, Smart CD: Over-expression of snakin-2 and extensin-like protein genes restricts pathogen invasiveness and enhances tolerance to Clavibacter michiganensis subsp. michiganensis in transgenic tomato (Solanum lycopersicum). Transgenic Res. 2012, 21 (1): 23-37. 10.1007/s11248-011-9506-x.View ArticlePubMedGoogle Scholar
- Kovalskaya N, Zhao Y, Hammond RW: Antibacterial and antifungal activity of a snakin-defensin hybrid protein expressed in tobacco and potato plant. Open Plant Sci J. 2011, 5: 29-42. 10.2174/1874294701105010029.View ArticleGoogle Scholar
- Balaji V, Sessa G, Smart CD: Silencing of host basal defense response-related gene expression increases susceptibility of Nicotiana benthamiana to Clavibacter michiganensis subsp. michiganensis. Phytopathology. 2011, 101 (3): 349-357. 10.1094/PHYTO-05-10-0132.View ArticlePubMedGoogle Scholar
- Nahirñak V, Almasia NI, Fernandez PV, Hopp HE, Estevez JM, Carrari F, Vazquez-Rovere C: Potato snakin-1 gene silencing affects cell division, primary metabolism, and cell wall composition. Plant Physiol. 2012, 158 (1): 252-263. 10.1104/pp.111.186544.PubMed CentralView ArticlePubMedGoogle Scholar
- Nahirñak V, Almasia NI, Hopp HE, Vazquez-Rovere C: Snakin/GASA proteins: involvement in hormone crosstalk and redox homeostasis. Plant Signal Behav. 2012, 7 (8): 1004-1008. 10.4161/psb.20813.PubMed CentralView ArticlePubMedGoogle Scholar
- Porto WF, Franco OL: Theoretical structural insights into the snakin/GASA family. Peptides. 2013, 44: 163-167. 10.1016/j.peptides.2013.03.014.View ArticlePubMedGoogle Scholar
- Howell CR, Stipanovic RD: Control of Rhizoctonia solani on cotton seedlings with Pseudomonas fluorescens with an antibiotic produced by the bacterium. Phytopathology. 1979, 69: 480-482. 10.1094/Phyto-69-480.View ArticleGoogle Scholar
- Carelli M, Gnocchi S, Fancelli S, Mengoni A, Paffetti D, Scotti C, Bazzicalupo M: Genetic diversity and dynamics of Sinorhizobium meliloti populations nodulating different alfalfa cultivars in Italian soils. Appl Environ Microbiol. 2000, 66 (11): 4785-4789. 10.1128/AEM.66.11.4785-4789.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Selbitschka W, Keller M, Miethling-Graff R, Dresing U, Schwieger F, Krahn I, Homann I, Dammann-Kalinowski T, Puhler A, Tebbe CC: Long-term field release of bioluminescent Sinorhizobium meliloti strains to assess the influence of a recA mutation on the strains’ survival. Microb Ecol. 2006, 52 (3): 583-595. 10.1007/s00248-006-9056-6.View ArticlePubMedGoogle Scholar
- Reeve W, Chain P, O’Hara G, Ardley J, Nandesena K, Brau L, Tiwari R, Malfatti S, Kiss H, Lapidus A, Copeland A, Nolan M, Land M, Hauser L, Chang YJ, Ivanova N, Mavromatis K, Markowitz V, Kyrpides N, Gollagher M, Yates R, Dilworth M, Howieson J: Complete genome sequence of the Medicago microsymbiont Ensifer (Sinorhizobium) medicae strain WSM419. Stand Genomic Sci. 2010, 2 (1): 77-86. 10.4056/sigs.43526.PubMed CentralView ArticlePubMedGoogle Scholar
- Keyser HH, Bohlool BB, Hu TS, Weber DF: Fast-growing rhizobia isolated from root nodules of soybean. Science. 1982, 215 (4540): 1631-1632. 10.1126/science.215.4540.1631.View ArticlePubMedGoogle Scholar
- Eardly BD, Hannaway DB, Bottomley PJ: Characterization of Rhizobia from ineffective Alfalfa nodules: ability to nodulate bean plants [Phaseolus vulgaris (L.) Savi.]. Appl Environ Microbiol. 1985, 50 (6): 1422-1427.PubMed CentralPubMedGoogle Scholar
- Segovia L, Young JP, Martinez-Romero E: Reclassification of American Rhizobium leguminosarum biovar phaseoli type I strains as Rhizobium etli sp. nov. Int J Syst Bacteriol. 1993, 43 (2): 374-377. 10.1099/00207713-43-2-374.View ArticlePubMedGoogle Scholar
- Kaneko T, Nakamura Y, Sato S, Asamizu E, Kato T, Sasamoto S, Watanabe A, Idesawa K, Ishikawa A, Kawashima K, Kimura T, Kishida Y, Kiyokawa C, Kohara M, Matsumoto M, Matsuno A, Mochizuki Y, Nakayama S, Nakazaki N, Shimpo S, Sugimoto M, Takeuchi C, Yamada M, Tabata S: Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti (supplement). DNA Res. 2000, 7 (6): 381-406. 10.1093/dnares/7.6.381.View ArticlePubMedGoogle Scholar
- Mathis JN, Israel DW, Barbour WM, Jarvis BD, Elkan GH: Analysis of the symbiotic performance of bradyrhizobium japonicum USDA 110 and its derivative I-110 and discovery of a new mannitol-utilizing, nitrogen-fixing USDA 110 derivative. Appl Environ Microbiol. 1986, 52 (1): 75-80.PubMed CentralPubMedGoogle Scholar
- Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA: A binary plant vector strategy based on separation of vir- and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature. 1983, 303 (5913): 179-180. 10.1038/303179a0.View ArticleGoogle Scholar
- Setten L, Soto G, Mozzicafreddo M, Fox AR, Lisi C, Cuccioloni M, Angeletti M, Pagano E, Diaz-Paleo A, Ayub ND: Engineering Pseudomonas protegens Pf-5 for nitrogen fixation and its application to improve plant growth under nitrogen-deficient conditions. PLoS One 2013, 8(5):e63666.,PubMed CentralView ArticlePubMedGoogle Scholar
- Ayub ND, Julia Pettinari M, Mendez BS, Lopez NI: Impaired polyhydroxybutyrate biosynthesis from glucose in Pseudomonas sp. 14–3 is due to a defective beta-ketothiolase gene. FEMS Microbiol Lett. 2006, 264 (1): 125-131. 10.1111/j.1574-6968.2006.00446.x.View ArticlePubMedGoogle Scholar
- Gleave AP: A versatile binary vector system with a T-DNA organisational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol. 1992, 20 (6): 1203-1207. 10.1007/BF00028910.View ArticlePubMedGoogle Scholar
- Campanella JJ, Bitincka L, Smalley J: MatGAT: an application that generates similarity/identity matrices using protein or DNA sequences. BMC Bioinformatics 2003, 4:29.,PubMed CentralView ArticlePubMedGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28 (10): 2731-2739. 10.1093/molbev/msr121.PubMed CentralView ArticlePubMedGoogle Scholar
- Finn RD, Mistry J, Tate J, Coggill P, Heger A, Pollington JE, Gavin OL, Gunasekaran P, Ceric G, Forslund K, Holm L, Sonnhammer EL, Eddy SR: The Pfam protein families database. Nucleic Acids Res. 2010, 38 (Database issue): D211-D222. 10.1093/nar/gkp985.PubMed CentralView ArticlePubMedGoogle Scholar
- Shen HB, Chou KC: Signal-3 L: a 3-layer approach for predicting signal peptides. Biochem Biophys Res Commun. 2007, 363 (2): 297-303. 10.1016/j.bbrc.2007.08.140.View ArticlePubMedGoogle Scholar
- Moller EM, Bahnweg G, Sandermann H, Geiger HH: A simple and efficient protocol for isolation of high molecular weight DNA from filamentous fungi, fruit bodies, and infected plant tissues. Nucleic Acids Res. 1992, 20 (22): 6115-6116. 10.1093/nar/20.22.6115.PubMed CentralView ArticlePubMedGoogle Scholar
- Ayub ND, Pettinari MJ, Ruiz JA, Lopez NI: A polyhydroxybutyrate-producing Pseudomonas sp. isolated from Antarctic environments with high stress resistance. Curr Microbiol. 2004, 49 (3): 170-174. 10.1007/s00284-004-4254-2.View ArticlePubMedGoogle Scholar
- Beringer JE: R factor transfer in Rhizobium leguminosarum. J Gen Microbiol. 1974, 84 (1): 188-198. 10.1099/00221287-84-1-188.PubMedGoogle Scholar
- Shen WJ, Forde BG: Efficient transformation of Agrobacterium spp. by high voltage electroporation. Nucleic Acids Res 1989, 17(20):8385.,PubMed CentralView ArticlePubMedGoogle Scholar
- D’Halluin K, Botterman J, De Greef W: Engineering of herbicide-resistant Alfalfa and evaluation under field conditions. Crop Sci. 1990, 30: 866-871. 10.2135/cropsci1990.0011183X003000040020x.View ArticleGoogle Scholar
- Soto G, Fox R, Ayub N, Alleva K, Guaimas F, Erijman EJ, Mazzella A, Amodeo G, Muschietti J: TIP5;1 is an aquaporin specifically targeted to pollen mitochondria and is probably involved in nitrogen remobilization in Arabidopsis thaliana. Plant J. 2010, 64 (6): 1038-1047. 10.1111/j.1365-313X.2010.04395.x.View ArticlePubMedGoogle Scholar
- Sanchez L, Weidmann S, Arnould C, Bernard AR, Gianinazzi S, Gianinazzi-Pearson V: Pseudomonas fluorescens and Glomus mosseae trigger DMI3-dependent activation of genes related to a signal transduction pathway in roots of Medicago truncatula. Plant Physiol. 2005, 139 (2): 1065-1077. 10.1104/pp.105.067603.PubMed CentralView ArticlePubMedGoogle Scholar
- Perez Di Giorgio J, Soto G, Alleva K, Jozefkowicz C, Amodeo G, Muschietti JP, Ayub ND: Prediction of aquaporin function by integrating evolutionary and functional analyses. J Membr Biol. 2014, 247 (2): 107-125. 10.1007/s00232-013-9618-8.View ArticlePubMedGoogle Scholar
- Furukawa T, Sakaguchi N, Shimada H: Two OsGASR genes, rice GAST homologue genes that are abundant in proliferating tissues, show different expression patterns in developing panicles. Genes Genet Syst. 2006, 81: 171-180. 10.1266/ggs.81.171.View ArticlePubMedGoogle Scholar
- Roxrud I, Lid SE, Fletcher JC, Schmidt ED, Opsahl-Sorteberg HG: GASA4, one of the 14-member Arabidopsis GASA family of small polypeptides, regulates flowering and seed development. Plant Cell Physiol. 2007, 48 (3): 471-483. 10.1093/pcp/pcm016.View ArticlePubMedGoogle Scholar
- Bennici A: Origin and early evolution of land plants: Problems and considerations. Commun Integr Biol. 2008, 1 (2): 212-218. 10.4161/cib.1.2.6987.PubMed CentralView ArticlePubMedGoogle Scholar
- Choi HK, Kim D, Uhm T, Limpens E, Lim H, Mun JH, Kalo P, Penmetsa RV, Seres A, Kulikova O, Roe BA, Bisseling T, Kiss GB, Cook DR: A sequence-based genetic map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics. 2004, 166 (3): 1463-1502. 10.1534/genetics.166.3.1463.PubMed CentralView ArticlePubMedGoogle Scholar
- Cook DR: Medicago truncatula–a model in the making!. Curr Opin Plant Biol. 1999, 2 (4): 301-304. 10.1016/S1369-5266(99)80053-3.View ArticlePubMedGoogle Scholar
- McCann MC, Rogan GJ, Fitzpatrick S, Trujillo WA, Sorbet R, Hartnell GF, Riodan SG, Nemeth MA: Glyphosate-tolerant alfalfa is compositionally equivalent to conventional alfalfa (Medicago sativa L.). J Agr Food Chem. 2006, 54 (19): 7187-7192. 10.1021/jf061482m.View ArticleGoogle Scholar
- Masoud SA, Zhu Q, Lamb C, Dixon RA: Constitutive expression of an inducible glucanase in alfalfa reduces disease severity caused by the oomycete pathogen Phytophthora megasperma f. sp medicaginis, but does not reduce disease severity of chitin-containing fungi. Transgenic Res. 1996, 5: 313-323. 10.1007/BF01968941.View ArticleGoogle Scholar
- Hipskind JD, Paiva NL: Constitutive accumulation of a resveratrol-glucoside in transgenic alfalfa increases resistance to Phoma medicaginis. Mol Plant Microbe Interact. 2000, 13 (5): 551-562. 10.1094/MPMI.2000.13.5.551.View ArticlePubMedGoogle Scholar
- Gruenheid S, Le Moual H: Resistance to antimicrobial peptides in Gram-negative bacteria. FEMS Microbiol Lett. 2012, 330 (2): 81-89. 10.1111/j.1574-6968.2012.02528.x.View ArticlePubMedGoogle Scholar
- Doyle JJ: Phylogenetic perspectives on the origins of nodulation. Mol Plant Microbe Interact. 2011, 24 (11): 1289-1295. 10.1094/MPMI-05-11-0114.View ArticlePubMedGoogle Scholar
- Jones KM, Kobayashi H, Davies BW, Taga ME, Walker GC: How rhizobial symbionts invade plants: the Sinorhizobium-Medicago model. Nat Rev Microbiol. 2007, 5 (8): 619-633. 10.1038/nrmicro1705.PubMed CentralView ArticlePubMedGoogle Scholar
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