Daria Shumilina, Ksenia Kromina, Vitaly Dzhavakhiya
All Russian Research Institute of Phytopathology
Golitsyno, Moscow region, 143050, Russia
Series of bacterial proteins (MF-proteins) were discovered, isolated and characterized as inductors a broad-spectrum resistance to viral, bacterial and fungal pathogens in plants, and thus constituted an alternative approach to protection crops from these pathogens. These proteins increase the resistance of various crops to pathogens when are used as pesticides (e.g. potato plants- against viral, bacterial and fungal pathogens; wheat plants- against septoriosis, cabbage and tobacco plants- against viral pathogens). Increasing in resistance observed is substantial, and the proteins are effective in very small amounts. After determination of MF genes structure, it was revealed, that one elicitor (MF2) belonged to cold shock proteins family, another one (MF3) has shown to possess high level of homology with peptidyl-prolyl cis-trans isomerases of FKBP-type. It was shown MF-proteins had no phytotoxic effect on plants. The amino acid sequences of MF‑proteins, DNA sequences of the mf- genes and their biological activities were covered by International patents.
Introduction
One of the most promising ways to search for highly effective methods for plant protection from pathogens and pests is the use of genetically incorporated plant potential. Understanding that plant resistance is a result of certain biochemical mechanisms is the base for development of such methods. In some cases, it is possible to solve a problem of yield losses caused by phytopathogens using specific regulators influencing biochemical reactions in plant tissues. It is well known that some rhizosphere or phyllosphere bacteria such as Pseudomonas fluorescens or Bacillus thuringiensis are successfully used as biological control agents or plant growth stimulators since alive bacteria can enhance plant growth and protect crops against pathogens via different mechanisms including induction of resistance in plants.
As a result of various microorganism screening for ability to induce defensive reactions in plant, extracts from B. thuringiensis and P. fluorescens (strain 197) were found to induce resistance of tobacco leaves (Nicotiana tabacum cv. Xanthi NN) against tobacco mosaic virus (TMV). Afterwards, it was determined that active substances of bacterial extracts were low-molecular (7.239 and 16.929 kDa) thermostable proteins named correspondingly Microbial Factor 2 (MF2) and Microbial Factor 3 (MF3). After determination of both elicitor proteins gene structures, it was revealed, that there were found two proteins with unrelated structure. Aligning amino acid sequences of elicitors with known sequences in GenBank it was found that one elicitor (MF2) belongs to cold shock proteins family. It s sequence was deposited to GenBank as Cold shock protein D (CspD). Another one (MF3) has shown to possess high level of homology with peptidyl-prolyl cis-trans isomerases of FKBP-type.
CspD (MF2 from B. thuringiensis)
CspD from Bacillus thuringiensis induced wide range resistance in plants to pathogens. The elicitor properties of CspD were studied in followed pairs plant – pathogen: tobacco - Tobacco Mosaic Virus (TMV) and tobacco - Potato Virus X (PVX); potato - late blight disease (Phytophthora infestans); rice - rice blast disease Magnaporthe grisea (Pyricularia oryzae); wheat – Septoria nodorum and wheat – brown rust.
Peptide from conservative region of CSPs
At first, it was shown that peptide consisted of only 15 amino acid residues (csp15) from the conservative region of bacterial cold shock proteins was able to induce accumulation of salicylic acid (an important component in plant defense signaling pathways) in potato. The concentration of salicylic acid in pretreated tubers was four times higher then in control. Secondly, we have shown that peptide csp15 possess protective activity in the following plant-pathogen pairs.
Potato – Phytophtora infestans
Slices of potato tubers were pretreated with peptide csp15. Next day the slices were inoculated with Ph. infestans. Pretreated potato tuber slices possessed an enhanced resistance against Ph. infestans causual agent of economically important late blight disease.
Wheat - Stagonospora nodorum (Septoria nodorum)
Wheat leaves (cv. Mironovskaya 808) were treated with peptide csp15. After 24 hours they were inoculated with S. nodorum spore suspension. The disease symptoms were watched for the next 7 days. It was shown that disease development on leaves pretreated with csp15 was significantly lower than on control leaves.
Tobacco – TMV
Tobacco leaves (cv. Xanthi NN) were pretreated with different concentration of peptide csp15. Next day the leaves were inoculated with TMV. Three days later, the number of invective lesions was scored. The number of TMV lesions developed on treated leaves was 50 % less then on control leaves.
MF3 protein from P. fluorescens
Bacterial strain – super producer of MF3 protein
To provide large scale production the protein expression vector (pMF) was created by replacement of a gene 10 in a vector pGEMEX1 on mf3 gene. To produce MF3, the plasmid DNA of pMF was transformed into E. coli strains BL21 (DE3). Thus, the subsequent tests were carried out using MF3-protein pure preparation. The ability of MF3-protein to induce non specific resistance in plants against pathogens was shown using the different plant - pathogen pairs.
Potato – Phytophtora infestans
Potato plants treated with MF3 were less sensitive to P. infestans. The decrease in number of lesions per cm2 leaf surface formed by P. infestans on plants treated with protein was around 70% in comparison with control plants. Diameter of lesions and amount of P. infestans spores (103) per lesion on leaves treated with MF3 were around 88% and 75% lower then in control.
Wheat - Stagonospora nodorum (Septoria nodorum)
Wheat leaves (cv. Mironovskaya 808) were treated with MF3-protein. After 24 hours they were inoculated with S. nodorum spore suspension. The disease symptoms were watched for next 7 days. It was shown that disease development on leaves pretreated with MF3 was significantly lower than on control leaves.
Rice - Magnaporthe grisea (Pyricularia oryzae)
We showed that rice pathogen M. grisea did not infect leaves of rice (cv. Sha-Tiao-Tsao) treated with MF3.
Tobacco - Alternaria longipes
Treatment with MF3 also induced the resistance of tobacco leaves (cv. Xanthy NN) against A. longipes. (Fig.I)
Figure I. Development of A. longipes on leaves of tobacco pretreated with MF3-protein and water.
Tobacco – TMV
Tobacco plants treated with low concentration of MF3-protein were not infected by TMV. Resistance to TMV infection was kept at least within 3 weeks. Spraying of only several leaves tobacco plant (cv. Samsun nn) was sufficient in order to induce systemic resistance of whole plant (Fig. II). Probably, MF3 capable of interacting with receptors on surface of plants or (and) penetrate into tissue across stomas.
Figure II. Dynamics of TMV development in tobacco plants (cv. Samsun nn).
White cabbage - turnip mosaic virus (TuMV)
Experiment with white cabbage showed that juice of leaves treated with MF3 contained lower concentration of TuMV as compared to control leave juice.
Transgenic potato - Erwinia carotovora and potato virus X
Another way for studying of MF3 defense inducing properties was creation of transgenic plants carrying mf3 gene. Selected transgenic plants expressing MF3-protein had normal phenotype and fertility. Tests of transgenic potato tubers showed that they are more resistant to Erwinia carotovora than tubers of non transgenic plants. Furthermore, transgenic plants of potato showed tolerance to potato virus X
Determination of MF3 protein active part.
It was hypothesized that active site of MF3 might be a part of conservative region of the protein. For determine active part of MF3, the protein was digested with trypsin that cut MF3 at a region of FKBP-type peptidyl-prolyl cis-trans isomerase signature 2. The enzymatic hydrolisis leaded to the loss of elicitor activity. Then, based on these data, 29 amino acid peptide was synthesized that was shown to retain the protective properties.
Conclusion
Obtained results suggest that CspD and MF3 proteins can be used for development of new bio-pesticides since it has a number of advantages. First, MF-proteins have no phytotoxic effect on plants. Second - it has no direct negative effects on the pathogens. MF- proteins influence on pathogens only across plants defense system. It allows avoiding situations with pathogen mutations and appearance of MF-resistant strains. Third, MF-proteins induce nonspecific resistance against viral, bacterial and fungal pathogens both in monocotyledons and dicotyledons plants. Next, practical application of MF‑protein can be many-sided, including production of transgenic plants carrying mf genes resistant to phytopathogens. Moreover, it is possible to use MF-proteins as bio-pesticides for treatment plants by spraying. With using methods of molecular biology bacterial strain – super producer of MF3 protein was constructed. Industrial schemes for synthesis, isolation and purification of MF3 were developed. It is important that MF-proteins have active parts which can be synthesized and used as bio-pesticide also. MF3-protein and mf3 gene structures, defense properties of protein were patented in Patent office of Finland by PCT International system: Dzhavakhia Vitaly, Shumilina Daria et al. “Proteins inducing multiple resistance of plants to phytopathogens and pests” WO2005061533 publication date 2005.07.07. The amino acid sequence of the MF2-protein and DNA sequence of the gene of this protein were covered by PCT International Patent Application (Publication number: WO 97/05165. International publication date: 13 February 1997; United States Patent No. 6,528,480 Djavakhia et al., March 4th 2003,"Protein with plant protecting properties").
Now the most internationally known company producing ecological pure bio-pesticide on base protein-inductors of natural defense reactions in plants is Eden Bioscience Corporation. Products of EDEN are based on naturally occurring proteins called “harpins”, which activate a plant’s intrinsic ability to protect itself through growth and stress-defense responses. Net product sales of EDEN for the fourth quarter of 2005 were $588,000, an increase of 250% when compared to net product sales of $168,000 in the same period of 2004. It is evidence of rapid development of bio-pesticides on base of proteins use.
MF-proteins have some advantages in comparison with harpins. They are low molecular weight, termostable and did not induce hypersensitive response (HR) in plants. Hence, MF-proteins have great chance to become new biological agents for the defense of crops.
Patents:
1. Djavakhia V. et al. "Protein with plant protecting properties" PCT International Patent Application (Publication number: WO 97/05165. International publication date: 13 February 1997; United States Patent No. 6,528,480, March 4th 2003)
2. Dzhavakhia V. et al. “Proteins inducing multiple resistance of plants to phytopathogens and pests” WO2005061533 publication date 2005-07-07 to PCT International system to Finnish patent office