Pathogen lifestyle determines host genetic signature of quantitative disease resistance loci in oilseed rape (Brassica napus).

KEY MESSAGE: Using associative transcriptomics, our study identifies genes conferring resistance to four diverse fungal pathogens in crops, emphasizing key genetic determinants of multi-pathogen resistance. Crops are affected by several pathogens, but these are rarely studied in parallel to identify common and unique genetic factors controlling diseases. Broad-spectrum quantitative disease resistance (QDR) is desirable for crop breeding as it confers resistance to several pathogen species. Here, we use associative transcriptomics (AT) to identify candidate gene loci associated with Brassica napus constitutive QDR to four contrasting fungal pathogens: Alternaria brassicicola, Botrytis cinerea, Pyrenopeziza brassicae, and Verticillium longisporum. We did not identify any shared loci associated with broad-spectrum QDR to fungal pathogens with contrasting lifestyles. Instead, we observed QDR dependent on the lifestyle of the pathogen-hemibiotrophic and necrotrophic pathogens had distinct QDR responses and associated loci, including some loci associated with early immunity. Furthermore, we identify a genomic deletion associated with resistance to V. longisporum and potentially broad-spectrum QDR. This is the first time AT has been used for several pathosystems simultaneously to identify host genetic loci involved in broad-spectrum QDR. We highlight constitutive expressed candidate loci for broad-spectrum QDR with no antagonistic effects on susceptibility to the other pathogens studies as candidates for crop breeding. In conclusion, this study represents an advancement in our understanding of broad-spectrum QDR in B. napus and is a significant resource for the scientific community.

Leaf position-dependent effect of Alternaria brassicicola development on host cell death, photosynthesis and secondary metabolites in Brassica juncea.

During the first 24 hours of infection, Alternaria brassicicola developmental parameters such as conidial germination, germ tubes and appressoria formation on each of the five mature Brassica juncea leaves, correlated with a leaf position showing stronger development of the pathogen on older leaves than on young ones. As a consequence of fungal development, the black spot disease was observed during 96 hours of infection on a macroscopic scale, as well as via confocal microscopy. Degradation of the chloroplast thylakoids and plastoglobule appearance during infection, followed by the decrease in chlorophyll a fluorescence parameters i.e. maximum quantum yield of PSII (Fv /Fm ), non-photochemical quenching (NPQ) and chlorophyll a:b ratio, have been observed. Also, after an initial increase of carbohydrates (glucose, fructose and sucrose), content far below the respective control values was found. The content of secondary metabolites such as flavonoids and glucosinolates increased in a leaf position-dependent manner in infected leaves, with a lower level in older leaves than in younger ones. Although, the total phenolic compounds (TPCs) content did not differ significantly in infected leaves compared to control leaves, TPCs level in both control and infected leaves was leaf position-dependent. To the best of our knowledge, this is the first report on leaf position-dependent effect on the B. juncea biochemical response to A. brassicicola infection.

The pharmaceutics from the foreign empire: the molecular pharming of the prokaryotic staphylokinase in Arabidopsis thaliana plants.

Here, we present the application of microbiology and biotechnology for the production of recombinant pharmaceutical proteins in plant cells. To the best of our knowledge and belief it is one of few examples of the expression of the prokaryotic staphylokinase (SAK) in the eukaryotic system. Despite the tremendous progress made in the plant biotechnology, most of the heterologous proteins still accumulate to low concentrations in plant tissues. Therefore, the composition of expression cassettes to assure economically feasible level of protein production in plants remains crucial. The aim of our research was obtaining a high concentration of the bacterial anticoagulant factor-staphylokinase, in Arabidopsis thaliana seeds. The coding sequence of staphylokinase was placed under control of the ß-phaseolin promoter and cloned between the signal sequence of the seed storage protein 2S2 and the carboxy-terminal KDEL signal sequence. The engineered binary vector pATAG-sak was introduced into Arabidopsis thaliana plants via Agrobacterium tumefaciens-mediated transformation. Analysis of the subsequent generations of Arabidopsis seeds revealed both presence of the sak and nptII transgenes, and the SAK protein. Moreover, a plasminogen activator activity of staphylokinase was observed in the protein extracts from seeds, while such a reaction was not observed in the leaf extracts showing seed-specific activity of the ß-phaseolin promoter.

Phaseolus vulgaris - recalcitrant potential.

Since the ability to genetically engineer plants was established, researchers have modified a great number of plant species to satisfy agricultural, horticultural, industrial, medicinal or veterinary requirements. Almost thirty years after the first approaches to the genetic modification of pulse crops, it is possible to transform many grain legumes. However, one of the most important species for human nutrition, Phaseolus vulgaris, still lacks some practical tools for genomic research, such as routine genetic transformation. Its recalcitrance towards in vitro regeneration and rooting significantly hampers the possibilities of improvement of the common bean that suffers from many biotic and abiotic constraints. Thus, an efficient and reproducible system for regeneration of a whole plant is desired. Although noticeable progress has been made, the rate of recovery of transgenic lines is still low. Here, the current status of tissue culture and recent progress in transformation methodology are presented. Some major challenges and obstacles are discussed and some examples of their solutions are presented.

Elastin-like polypeptides as a promising family of genetically-engineered protein based polymers.

Elastin-like polypeptides (ELP) are artificial, genetically encodable biopolymers, belonging to elastomeric proteins, which are widespread in a wide range of living organisms. They are composed of a repeating pentapeptide sequence Val-Pro-Gly-Xaa-Gly, where the guest residue (Xaa) can be any naturally occurring amino acid except proline. These polymers undergo reversible phase transition that can be triggered by various environmental stimuli, such as temperature, pH or ionic strength. This behavior depends greatly on the molecular weight, concentration of ELP in the solution and composition of the amino acids constituting ELPs. At a temperature below the inverse transition temperature (Tt), ELPs are soluble, but insoluble when the temperature exceeds Tt. Furthermore, this feature is retained even when ELP is fused to the protein of interest. These unique properties make ELP very useful for a wide variety of biomedical applications (e.g. protein purification, drug delivery etc.) and it can be expected that smart biopolymers will play a significant role in the development of most new materials and technologies. Here we present the structure and properties of thermally responsive elastin-like polypeptides with a particular emphasis on biomedical and biotechnological application.

Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the ß-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

R proteins as fundamentals of plant innate immunity.

Plants are attacked by a wide spectrum of pathogens, being the targets of viruses, bacteria, fungi, protozoa, nematodes and insects. Over the course of their evolution, plants have developed numerous defense mechanisms including the chemical and physical barriers that are constitutive elements of plant cell responses locally and/or systemically. However, the modern approach in plant sciences focuses on the evolution and role of plant protein receptors corresponding to specific pathogen effectors. The recognition of an invader's molecules could be in most cases a prerequisite sine qua non for plant survival. Although the predicted three-dimensional structure of plant resistance proteins (R) is based on research on their animal homologs, advanced technologies in molecular biology and bioinformatics tools enable the investigation or prediction of interaction mechanisms for specific receptors with pathogen effectors. Most of the identified R proteins belong to the NBS-LRR family. The presence of other domains (including the TIR domain) apart from NBS and LRR is fundamental for the classification of R proteins into subclasses. Recently discovered additional domains (e.g. WRKY) of R proteins allowed the examination of their localization in plant cells and the role they play in signal transduction during the plant resistance response to biotic stress factors. This review focuses on the current state of knowledge about the NBS-LRR family of plant R proteins: their structure, function and evolution, and the role they play in plant innate immunity.

Expression of a staphylokinase, a thrombolytic agent in Arabidopsis thaliana.

A gene encoding staphylokinase from Staphylococcus aureus was cloned into the plant transformation binary vector pCAMBIA 1304. The transgene was introduced into the genome of A. thaliana via in planta Agrobacterium tumefaciens-mediated genetic transformation. The presence of the staphylokinase gene was confirmed by PCR in 60% of the investigated plants. The presence of the fusion protein (119 kDa) was confirmed by SDS-PAGE and Western blot analysis in protein extracts from putative transgenics. Furthermore, the amidolytic assay confirmed the activity of SAK in protein extracts in 23 out of 45 transgenic lines of A. thaliana plants.

The evaluation of the genotoxicity of two commonly used food colors: Quinoline Yellow (E 104) and Brilliant Black BN (E 151).

Additives, especially colors, are in widespread use in the food industry. With the exception of the quinolines, food colors are relatively weak mutagens and are certified as safe additives despite reports that some people have allergic reactions to them. The number of food additives is still on the increase, and research on their potential mutagenic/carcinogenic activity in vivo is very expensive. Using two different cellular model systems, human lymphocytes in vitro and Vicia faba root tip meristems of in vivo, we evaluated the potential cytological and genotoxic effects of two dyes: Quinoline Yellow (E 104) and Brilliant Black BN (E 151). Two relatively new, very sensitive and rapid tests - the micronucleus and Comet assays - were used in this study. The data provided in this paper showed the genotoxic effects of the two analyzed food colors, and confirmed the diagnostic value of the MN and Comet assays for screening potentially genotoxic substances.