Wheat Pore-Forming Toxin-Like Protein Confers Broad-Spectrum Resistance to Fungal Pathogens in Arabidopsis.

Fusarium head blight (FHB), caused by the hemibiotrophic fungus Fusarium graminearum, is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie Fhb1, the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant Arabidopsis. Heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. To explore the reason for the resistance response to, exclusively, the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or oomycete species. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytoplasma Infection Blocks Starch Breakdown and Triggers Chloroplast Degradation, Leading to Premature Leaf Senescence, Sucrose Reallocation, and Spatiotemporal Redistribution of Phytohormones.

Witches'-broom (WB, excessive initiation, and outgrowth of axillary buds) is one of the remarkable symptoms in plants caused by phytoplasmas, minute wall-less intracellular bacteria. In healthy plants, axillary bud initiation and outgrowth are regulated by an intricate interplay of nutrients (such as sugars), hormones, and environmental factors. However, how these factors are involved in the induction of WB by phytoplasma is poorly understood. We postulated that the WB symptom is a manifestation of the pathologically induced redistribution of sugar and phytohormones. Employing potato purple top phytoplasma and its alternative host tomato (Solanum lycopersicum), sugar metabolism and transportation, and the spatiotemporal distribution of phytohormones were investigated. A transmission electron microscopy (TEM) analysis revealed that starch breakdown was inhibited, resulting in the degradation of damaged chloroplasts, and in turn, premature leaf senescence. In the infected source leaves, two marker genes encoding asparagine synthetase (Sl-ASN) and trehalose-6-phosphate synthase (Sl-TPS) that induce early leaf senescence were significantly up-regulated. However, the key gibberellin biosynthesis gene that encodes ent-kaurene synthase (Sl-KS) was suppressed. The assessment of sugar content in various infected tissues (mature leaves, stems, roots, and leaf axils) indicated that sucrose transportation through phloem was impeded, leading to sucrose reallocation into the leaf axils. Excessive callose deposition and the resulting reduction in sieve pore size revealed by aniline blue staining and TEM provided additional evidence to support impaired sugar transport. In addition, a spatiotemporal distribution study of cytokinin and auxin using reporter lines detected a cytokinin signal in leaf axils where the axillary buds initiated. However, the auxin responsive signal was rarely present in such leaf axils, but at the tips of the newly elongated buds. These results suggested that redistributed sucrose as well as cytokinin in leaf axils triggered the axillary bud initiation, and auxin played a role in the bud elongation. The expression profiles of genes encoding squamosa promoter-binding proteins (Sl-SBP1), and BRANCHED1 (Sl-BRC1a and Sl-BRC1b) that control axillary bud release, as determined by quantitative reverse transcription (qRT)-PCR, indicated their roles in WB induction. However, their interactions with sugars and cytokinins require further study. Our findings provide a comprehensive insight into the mechanisms by which phytoplasmas induce WB along with leaf chlorosis, little leaf, and stunted growth.

Insights into the Transcriptional Reprogramming in Tomato Response to PSTVd Variants Using Network Approaches.

Viroids are the smallest pathogens of angiosperms, consisting of non-coding RNAs that cause severe diseases in agronomic crops. Symptoms associated with viroid infection are linked to developmental alterations due to genetic regulation. To understand the global mechanisms of host viroid response, we implemented network approaches to identify master transcription regulators and their differentially expressed targets in tomato infected with mild and severe variants of PSTVd. Our approach integrates root and leaf transcriptomic data, gene regulatory network analysis, and identification of affected biological processes. Our results reveal that specific bHLH, MYB, and ERF transcription factors regulate genes involved in molecular mechanisms underlying critical signaling pathways. Functional enrichment of regulons shows that bHLH-MTRs are linked to metabolism and plant defense, while MYB-MTRs are involved in signaling and hormone-related processes. Strikingly, a member of the bHLH-TF family has a specific potential role as a microprotein involved in the post-translational regulation of hormone signaling events. We found that ERF-MTRs are characteristic of severe symptoms, while ZNF-TF, tf3a-TF, BZIP-TFs, and NAC-TF act as unique MTRs. Altogether, our results lay a foundation for further research on the PSTVd and host genome interaction, providing evidence for identifying potential key genes that influence symptom development in tomato plants.

Development and optimization of a pepino mosaic virus-based vector for rapid expression of heterologous proteins in plants.

Plant-virus-derived vectors are versatile tools with multiple applications in agricultural and medical biotechnology. In this study, we developed pepino mosaic virus (PepMV) (family Alphaflexiviridae; genus Potexvirus) into a vector for heterologous protein expression in plants. PepMV was initially cloned in a step-wise manner, fully sequenced and the full-length infectious clone was tested for infectivity in Nicotiana benthamiana. Initial infectious clones resulted in poor replication of PepMV and lack of systemic movement. Mutations in the viral sequence affected systemic infection. Two suspected mutations were altered to restore systemic infectivity. PepMV infection was apparent as early as 4 days post agroinfiltration (dpa) inoculation in N. benthamiana. A multiple cloning site was inserted into the PepMV genome for introduction and expression of foreign genes. Several modifications to the wild-type vector were made, such as a replacing the native subgenomic promoter (SGP) with a heterologous SGP, and introduction of translational enhancers and terminators, to improve heterologous expression of the foreign gene-of-interest. GFP was used as a reporter for monitoring virus infection and protein production. Strong GFP expression was observed as early as 4 dpa with a translational enhancer. The PepMV-based vector produces rapid expression of the foreign gene in comparison to two other potexvirus-based vectors. GFP production was monitored over time and optimal protein production was recorded between 5 and 7 dpa. GFP protein levels reached up to 4% and decreased to 0.5% total soluble protein at 7 and 14 dpa, respectively. Future studies will evaluate this virus-based vector for large-scale production of pharmaceutical compounds. KEY POINTS: • A pepino mosaic virus isolate was developed into a plant-based expression vector. • Expression levels of the heterologous protein were comparable or exceeded previously developed viral vectors. • Protein levels in plants were highest between 5 and 7 days and decreased gradually.

Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression.

BACKGROUND: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains. RESULTS: An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100µg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue. CONCLUSION: We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

Complete nucleotide sequence of little cherry virus 1 (LChV-1) infecting sweet cherry in China.

Little cherry virus 1 (LChV-1), associated with little cherry disease (LCD), has a significant impact on fruit quality of infected sweet cherry trees. We report the full genome sequence of an isolate of LChV-1 from Taian, China (LChV-1-TA), detected by small-RNA deep sequencing and amplified by overlapping RT-PCR. The LChV-1-TA genome was 16,932 nt in length and contained nine open reading frames (ORFs), with sequence identity at the overall genome level of 76%, 76%, and 78% to LChV-1 isolates Y10237 (UW2 isolate), EU715989 (ITMAR isolate) and JX669615 (V2356 isolate), respectively. Based on the phylogenetic analysis of HSP70h amino acid sequences of Closteroviridae family members, LChV-1-TA was grouped into a well-supported cluster with the members of the genus Velarivirus and was also closely related to other LChV-1 isolates. This is the first report of the complete nucleotide sequence of LChV-1 infecting sweet cherry in China.

Yeast Expressing a Phage Endolysin Reduces Endogenous Clostridium perfringens Ex Vivo in 21-Day-Old Broiler Chicken Intestinal Fluids.

The phage endolysin PlyCP41 when purified from Escherichia coli exhibits lytic activity against Clostridium perfringens (CP) in vitro. The anti-clostridial activity of PlyCP41 endolysin expressed in transgenic yeast (Saccharomyces cerevisiae) was verified in phosphate buffered saline via mixing experiments with cultured CP and transgenic yeast slurries followed by serial dilution plating and colony counts on tryptose sulfite cycloserine (CP indicator) plates. The transgenic yeast containing PlyCP41 resulted in a log10 4.5 reduction (99.997%; P < 0.01) of the cultured CP. In addition, this serial dilution plating assay was used to demonstrate that transgenic yeast slurries could reduce the endogenous CP content in fluids from three different gastrointestinal regions (proximal, medial, and distal) from 21-day-old broiler chickens. The transgenic yeast treatment of gut slurries resulted in a log 10 1.19, 4.53, and 1.28 reduction in proximal, medial, and distal gut slurries (90% to 99.99% of the endogenous CP; P < 0.01), respectively, compared to nontreatment controls. These results indicate that the phage endolysin PlyCP41 expressed in S. cerevisiae is effective at reducing the endogenous CP in gastrointestinal fluids of broiler chickens. Future studies will measure the anti-CP effect in vivo by administering transgenic yeast to broiler chickens in the feed.

Levadura que expresa una fago-endolisina reduce la presencia endógena de Clostridium perfringens Ex vivo en fluidos intestinales de pollos de engorde de 21 días. La fago endolisina PlyCP41, cuando se purifica a partir de Escherichia coli, exhibe actividad lítica contra Clostridium perfringens (Cp) in vitro. La actividad anticlostridial de la endolisina PlyCP41 expresada en levadura transgénica (Saccharomyces cerevisiae) se verificó en solución salina amortiguada con fosfato mediante experimentos de mezclas con cultivos de C. perfringens y suspensiones de levadura transgénica, seguido de cultivos de diluciones en serie y recuentos de colonias en placas de triptosa sulfito cicloserina (TSC; indicador para C. perfringens). La levadura transgénica que contenía PlyCP41 dio como resultado una reducción de log10 4.5 (99.997%; P <0.01) en el cultivo de C. perfringens. Además, este ensayo de dilución en serie en placas se utilizó para demostrar que las suspensiones de levadura transgénica podrían reducir el contenido de C. perfringens endógeno en fluidos de tres regiones gastrointestinales diferentes (proximal, medial y distal) de pollos de engorde de 21 días de edad. El tratamiento con levadura transgénica de las suspensiones intestinales dio como resultado una reducción de log10 de 1.19, 4.53 y 1.28 en las suspensiones intestinales proximal, medial y distal (90% a 99.99 % de C. perfringens endógena; P < 0.01), respectivamente, en comparación con los controles no tratados. Estos resultados indican que la fago-endolisina PlyCP41 expresada en S. cerevisiae es eficaz para reducir el contenido endógeno de C. perfringens en los fluidos gastrointestinales de pollos de engorde. Los estudios futuros medirán el efecto contra C. perfringens in vivo mediante la administración de levadura transgénica a pollos de engorde en el alimento.

Coding-complete genome sequence of an isolate of papaya virus E in tomato.

An isolate of papaya virus E was identified in tomato fruits from Mexico. The coding-complete genome sequence was determined using high-throughput sequencing. The coding-complete genome is 13,412 nucleotides and contains 8 open reading frames.

Complete genome sequence of switchgrass mosaic virus, a member of a proposed new species in the genus Marafivirus.

The complete genome sequence of a virus recently detected in switchgrass (Panicum virgatum) was determined and found to be closely related to that of maize rayado fino virus (MRFV), genus Marafivirus, family Tymoviridae. The genomic RNA is 6408 nucleotides long. It contains three predicted open reading frames (ORFs 1-3), encoding proteins of 227 kDa, 43.9 kDa, and 31.5 kDa, compared to two ORFs (1 and 2) for MRFV. The complete genome shares 76 % sequence identity with MRFV. The nucleotide sequence of ORF2 of this virus and the amino acid sequence of its encoded protein are 49 % and 77 % identical, respectively, to those of MRFV. The virus-encoded polyprotein and capsid protein aa sequences are 83 % and 74-80 % identical, respectively, to those of MRFV. Although closely related to MRFV, the amino acid sequence of its capsid protein (CP) forms a clade that is separate from that of MRFV. Based on the International Committee on Taxonomy of Viruses (ICTV) sequence-related criteria for delineation of species within the genus Marafivirus, the virus qualifies as a member of a new species, and the name Switchgrass mosaic virus (SwMV) is proposed.

Detection and Characterization of Viroids via Biological Assays on Herbaceous Hosts.

The characterization of the elusive disease agent of the potato spindle tuber disease, potato spindle tuber viroid (PSTVd), was aided by the ability to obtain large amounts of infected tomato tissue in a simple bioassay where PSTVd was easily mechanically transmissible to an alternate herbaceous host in which it thrived and produced dramatic symptoms in a relatively short period (Diener, Viroids. Handbook of plant virus infections: comparative diagnosis. Elsevier/North-Holland, Amsterdam, pp 913-934, 1981; Diener, Virology 45:411-428, 1971; Raymer and O'Brien, Am Pot J, 39:401-408, 1962). Reactions in the primary, or secondary, herbaceous indicator host can range from asymptomatic to severe depending upon the viroid strain, host species, and environmental conditions and can provide evidence of a viroid infection, but do not permit identification of the viroid in question. Further characterization by molecular hybridization, RT-PCR, and sequence analysis is used to determine the etiology of the disease agent. In this chapter, methods are described for mechanical inoculation of viroids to herbaceous hosts to determine the viroid nature of diseases and the experimental host range of the viroid or to shorten the time required for obtaining relatively large amounts of viroid for subsequent purification and characterization.

Extraction and Purification of Viroids from Herbaceous Hosts.

Protocols for extraction and purification of viroid RNAs from the tissues of infected herbaceous plant hosts are numerous. They range from lengthy, traditional protocols that require large amounts of starting tissue and take several days to perform to those based on column chromatography which is more efficient and can be performed with smaller amounts of infected tissue. The goal of all protocols is to enrich for RNA fractions that contain viroid RNAs, and the RNA extraction procedure is chosen and adjusted for the downstream method used for detection and characterization. Removal of inhibitors/impurities is generally not an issue for herbaceous hosts unless they contain and inordinate amounts of polysaccharides, tannins, and phenols. Subsequent purification of viroid circular and linear RNAs is performed using denaturing polyacrylamide gel electrophoresis. In this chapter, a specific method routinely used for viroid purification from herbaceous hosts and problems that may be encountered is described and is intended as a reference for beginners in the field.

Cloning and Sequencing of Viroids.

Determining the sequence identity of viroid RNAs present in symptomatic or asymptomatic plant tissues is critical to obtain knowledge of their distribution. It enables the development of tools for diagnostics and for studying the basic biology of viroids. With the advent of cDNA-based methods for cloning RNAs and cloning strategies that do not require prior knowledge of the viroid sequence, characterization of several newly discovered viroids has rapidly expanded our knowledge of these unusual pathogenic RNAs. This chapter describes two methods, using random primers or viroid-specific primers, to generate complementary DNA (cDNA) copies of viroid RNAs for subsequent cloning and sequence analysis.

Rapid diagnostic detection of tomato apical stunt viroid based on isothermal reverse transcription-recombinase polymerase amplification.

Tomato apical stunt viroid (TASVd) is a serious threat to tomato plants that can cause a considerable yield loss. In the present study, two isothermal molecular diagnostic assays based on reverse transcription-recombinase polymerase amplification (RT-RPA) utilizing the AmplifyRP® platform for plant pathogen detection were developed. The results of this research demonstrated distinct specificity of both developed assays, AmplifyRP® Acceler8™ and AmplifyRP® XRT, expressed in the absence of any cross-reaction activity to all total RNA extracts obtained from plants infected with other pospiviroids. The RT-RPA assays detected viroid RNA in 81- and 27-fold dilutions of the original TASVd-infected crude extract for AmplifyRP® Acceler8™ and AmplifyRP® XRT, respectively. The sensitivity tests in serial water dilutions showed the ability of AmplifyRP® Acceler8™ and AmplifyRP® XRT to detect 8 and 80 fg of pure TASVd RNA transcript, respectively. The influence of crude extract on viroid RNA transcript detection was also examined and a decrease of sensitivity of approximately 100-fold for both RT-RPA assays was revealed. To our knowledge, this is the first report describing development of RT-RPA assays to detect TASVd in plants using the AmplifyRP® platform that can be further employed both in laboratory conditions and in the field for on-site diagnosis.

Mexico: A Landscape of Viroid Origin and Epidemiological Relevance of Endemic Species.

Viroids are single-stranded, circular RNA molecules (234-406 nt) that infect a wide range of crop species and cause economic losses in agriculture worldwide. They are characterized by the existence of a population of sequence variants, attributed to the low fidelity of RNA polymerases involved in their transcription, resulting in high mutation rates. Therefore, these biological entities exist as quasispecies. This feature allows them to replicate within a wide range of host plants, both monocots and dicots. Viroid hosts include economically important crops such as tomato, citrus, and fruit trees such as peach and avocado. Given the high risk of introducing viroids to viroid disease-free countries, these pathogens have been quarantined globally. As discussed herein, Mexico represents a geographical landscape of viroids linked to their origin and comprises considerable biodiversity. The biological features of viroid species endemic to Mexico are highlighted in this communication. In addition, we report the phylogenetic relationships among viroid and viroid strains, their economic impact, geographical distribution, and epidemiological features, including a broad host range and possible long-distance, seed, or insect-mediated transmission. In summary, this review could be helpful for a better understanding of the biology of viroid diseases and future programs on control of movement and spread to avoid economic losses in agricultural industries.

Complete genomic sequence of a Tobacco rattle virus isolate from Michigan-grown potatoes.

Tobacco rattle virus (TRV) causes stem mottle on potato leaves and necrotic arcs and rings in potato tubers, known as corky ringspot disease. Recently, TRV was reported in Michigan potato tubers cv. FL1879 exhibiting corky ringspot disease. Sequence analysis of the RNA-1-encoded 16-kDa gene of the Michigan isolate, designated MI-1, revealed homology to TRV isolates from Florida and Washington. Here, we report the complete genomic sequence of RNA-1 (6,791 nt) and RNA-2 (3,685 nt) of TRV MI-1. RNA-1 is predicted to contain four open reading frames, and the genome structure and phylogenetic analyses of the RNA-1 nucleotide sequence revealed significant homologies to the known sequences of other TRV-1 isolates. The relationships based on the full-length nucleotide sequence were different from than those based on the 16-kDa gene encoded on genomic RNA-1 and reflect sequence variation within a 20-25-aa residue region of the 16-kDa protein. MI-1 RNA-2 is predicted to contain three ORFs, encoding the coat protein (CP), a 37.6-kDa protein (ORF 2b), and a 33.6-kDa protein (ORF 2c). In addition, it contains a region of similarity to the 3' terminus of RNA-1, including a truncated portion of the 16-kDa cistron. Phylogenetic analysis of RNA-2, based on a comparison of nucleotide sequences with other members of the genus Tobravirus, indicates that TRV MI-1 and other North American isolates cluster as a distinct group. TRV M1-1 is only the second North American isolate for which there is a complete sequence of the genome, and it is distinct from the North American isolate TRV ORY. The relationship of the TRV MI-1 isolate to other tobravirus isolates is discussed.

Plant Virus-Derived Vectors: Applications in Agricultural and Medical Biotechnology.

Major advances in our understanding of plant viral genome expression strategies and the interaction of a virus with its host for replication and movement, induction of disease, and resistance responses have been made through the generation of infectious molecules from cloned viral sequences. Autonomously replicating viral vectors derived from infectious clones have been exploited to express foreign genes in plants. Applications of virus-based vectors include the production of human/animal therapeutic proteins in plant cells and the specific study of plant biochemical processes, including those that confer resistance to pathogens. Additionally, virus-induced gene silencing, which is RNA mediated and triggered through homology-dependent RNA degradation mechanisms, has been exploited as an efficient method to study the functions of host genes in plants and to deliver small RNAs to insects. New and exciting strategies for vector engineering, delivery, and applications of plant virus-based vectors are the subject of this review.

Complete Genome Sequence of an American Isolate of Pepino Mosaic Virus.

Pepino mosaic virus (PepMV) is a widely distributed tomato virus. The complete genome sequence of the PepMV isolate US3 from infected tomato fruit was determined. The genome is 6,410 nucleotides long and has a poly(A) tail. US3 shares the highest similarity with strains belonging to the European genotype.

Identification and characterization of Miscanthus yellow fleck virus, a new polerovirus infecting Miscanthus sinensis.

Miscanthus sinensis is a grass used for sugarcane breeding and bioenergy production. Using high throughput sequencing technologies, we identified a new viral genome in infected M. sinensis leaf tissue displaying yellow fleck symptoms. This virus is most related to members of the genus Polerovirus in the family Luteoviridae. The canonical ORFs were computationally identified, the P3 coat protein was expressed, and virus-like particles were purified and found to conform to icosahedral shapes, characteristic of the family Luteoviridae. We propose the name Miscanthus yellow fleck virus for this new virus.

Modification of tobacco plant development by sense and antisense expression of the tomato viroid-induced AGC VIIIa protein kinase PKV suggests involvement in gibberellin signaling.

BACKGROUND: The serine-threonine protein kinase gene, designated pkv (protein kinase- viroid induced) was previously found to be transcriptionally activated in tomato plants infected with the plant pathogen Potato spindle tuber viroid (PSTVd). These plants exhibited symptoms of stunting, and abnormal development of leaf, root, and vascular tissues. The encoded protein, PKV, is a novel member of the AGC VIIIa group of signal-transducing protein kinases; however, the role of PKV in plant development is unknown. In this communication, we report the phenotypic results of over expression and silencing of pkv in transgenic tobacco. RESULTS: Over expression of pkv in Nicotiana tabacum cv. Xanthi (tobacco) resulted in stunting, reduced root formation, and delay in flowering, phenotypes similar to symptoms of PSTVd infection of tomato. In addition, homozygous T2 tobacco plants over expressing PKV were male sterile. Antisense expression of pkv, on the other hand, resulted in plants that were taller than non-transformed plants, produced an increased number of flowers, and were fertile. Exogenous application of GA3 stimulated stem elongation in the stunted, sense-expressing plants. PKV sense and antisense expression altered transcript levels of GA biosynthetic genes and genes involved in developmental and signaling pathways, but not genes involved in salicylic acid- or jasmonic acid-dependent pathways. Our data provide evidence suggesting that PKV plays an important role in a GA signaling pathway that controls plant height and fertility. CONCLUSION: We have found that the over expression of the tomato protein kinase PKV resulted in stunting, modified vascular tissue development, reduced root formation, and male sterility in tobacco, and we propose that PKV regulates plant development by functioning in critical signaling pathways involved in gibberellic acid metabolism.

Expression and functional characterization of the plant antimicrobial snakin-1 and defensin recombinant proteins.

In this study, for the first time, functionally active, recombinant, cysteine-rich plant proteins snakin-1 (SN1) and defensin (PTH1) were expressed and purified using a prokaryotic expression system. The overall level of antimicrobial activities of SN1 and PTH1 produced in Escherichia coli was commensurate with that of the same proteins previously obtained from plant tissues. Both proteins exhibited strong antibacterial activity against the phytopathogenic bacterium Clavibacter michiganensis subsp. sepedonicus (50% inhibitory concentration (IC(50)) 1.5-8 microM) and antifungal activity against the phytopathogenic fungi Colletotrichum coccoides and Botrytis cinerea (IC(50) 5-14 microM). Significantly weaker activity was observed against Pseudomonas syringae pv. syringae and Pseudomonas syringae pv. tabaci. A pronounced synergistic antimicrobial effect against P. syringae pv. syringae and an additive effect against P. syringae pv. tabaci occurred with a combination of SN1 and PTH1. Aggregation of C. michiganensis subsp. sepedonicus bacterial cells at all protein concentrations tested was observed with the combination of SN1 and PTH1 and with SN1 alone. Our results demonstrate the use of a cost effective prokaryotic expression system for generation and in vitro characterization of plant cysteine-rich proteins with potential antimicrobial activities against a wide range of phytopathogenic microorganisms in order to select the most effective agents for future in vivo studies.