Jeandet, P.; Clément, C.; Courot, E.; Cordelier, S. Modulation of Phytoalexin Biosynthesis in Engineered Plants for Disease Resistance. Int. J. Mol. Sci.2013, 14, 14136-14170.
Abstract
Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms. Starting in the 1950’s, research on phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of phytoalexins has been investigated to increase the disease resistance of plants. The first example of a disease resistance resulting from foreign phytoalexin expression in a novel plant has concerned a phytoalexin from grapevine which was transferred to tobacco. Transformations were then operated to investigate the potential of other phytoalexin biosynthetic genes to confer resistance to pathogens. Unexpectedly, engineering phytoalexins for disease resistance in plants seem to have been limited to exploiting only a few phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. Research has rather focused on indirect approaches which allow modulation of the accumulation of phytoalexin employing transcriptional regulators or components of upstream regulatory pathways. Genetic approaches using gain- or less-of functions in phytoalexin engineering together with modulation of phytoalexin accumulation through molecular engineering of plant hormones and defense-related marker and elicitor genes have been reviewed.
Conclusions
Starting in the 1950’s, research on phytoalexins has begun with biochemistry and bio-organic chemistry, resulting in the determination of their structure, their biological activity as well as mechanisms of their synthesis and their catabolism by microorganisms. Elucidation of the biosynthesis of numerous phytoalexins has permitted the use of molecular biology tools for the exploration of the genes encoding enzymes of their synthesis pathways and their regulators. Genetic manipulation of phytoalexins requires a priori a sound knowledge of the genes involved in their biosynthesis and how accumulation of a given phytoalexin can be modulated. Success of the transformation will also depend on the inability of the pathogen to counteract the phytoalexin action. It is well known that phytopathogenic fungi, particularly, are able to metabolize the phytoalexins to which they are exposed. Engineering of fungal genes responsible for detoxification of phytoalexins in plants has pointed out their role in the interactions between plants and pathogens. For example, overexpression in hairy roots of pea (Pisum sativum L.) of a pisatin demethylating activity (PDA) from the pea-pathogen fungus Nectria haematococca reduced the amounts of pisatin. As a result, transgenic plant tissues with reduced capability to accumulate pisatin were found to be less resistant to fungal infection [85]. Also, it appears clearly evident that, in phytopathogenetic fungi, ATP-binding cassette (ABC) transporters, which may extrude plant defense products as well as fungicides, act as virulence factors, providing protection against defense compounds produced by the host. Many factors thus interplay which could affect the outcome of the interaction between plants and pathogens.
Unexpectedly, engineering phytoalexins for disease resistance in plants seems to have been limited to exploiting only a few phytoalexin biosynthetic genes, especially those encoding stilbenes and some isoflavonoids. The first example of a disease resistance resulting from foreign phytoalexin expression in a novel plant was published only in 1993 [65]. One can imagine that such a success would have opened the way for a ferment of activity in this area, but, if transformations were then operated to investigate the potential of stilbene biosynthetic genes to confer resistance to pathogens [59,60,62], strategy in engineering phytoalexins from other plant species did not receive as much applications as might be expected. As previously stated in this review, interest in secondary metabolite engineering deals with their implications in human health and disease. The extraordinary success obtained with resveratrol, the phytoalexin of Vitaceae, is linked to the fact that in this case, the engineered system requires a relatively simple genetic construct. Resveratrol is indeed obtained in one single step from p-coumaroyl-CoA and three malonyl-CoA units catalyzed by stilbene synthase. As these substrate precursor molecules are present throughout the plant kingdom, the introduction of a single gene is therefore sufficient to synthesize resveratrol in heterologous plant species. Except the case of resveratrol biosynthesis which appears to be very simple, other phytoalexins (isoflavonoids, terpenoids) are formed through very complex biosynthetic pathways. Engineering the entire pathway is not feasible and the problem is to choose the right enzyme catalyzing the limitant reaction of the given pathway. Moreover, modalities of the expression of genes encoding a given enzyme can be unpredictable. In fact, overexpression of 7-O-methyltransferase in alfalfa led to different regiospecificities, producing mainly 4′-O-methyl derivatives instead of the expected 7-O-methyl derivatives [83].
New techniques for metabolic engineering have thus to be exploited in the next years to come. Namely, methodologies for generating high-quality libraries of enzyme variants and novel high-throughput screening (HTS) technologies will open the way for the engineering of enzymes for the biosynthesis of various phytoalexins with potent biological activities. Specifically, HTS technologies can rapidly lead to the identification of genes which modulate a particular biosynthesis pathway. Gathering all genes encoding for a biomolecular pathway will allow the assembly of genetic constructs for the synthesis of a given phytoalexin.
Limitations in the strategy of engineering phytoalexins also arose from the fact that the obtained resistance is sometimes too weak since it is well known that phytoalexins are less phytotoxic than chemical fungicides [16]. This thus justifies indirect approaches which allow modulation of the accumulation of phytoalexin employing transcriptional regulators or components of upstream regulatory pathways (see above sections 4 and 5). For example, overexpression in Arabidopsis of the active MAPKs, MPK3/MPK6 activated the endogenous MAPKs, which in turn induced genes encoding the P450 enzymes operating in the biosynthetic pathway from tryptophan to camalexin, showing their activities a 50-fold increase or in some cases a 400-fold increase [13]. As a consequence, the data presented in this review show undoubtedly that indirect modulation of phytoalexin levels through transgenic approaches paves the way for the creation of novel plants with improved pathogen resistance traits.
See more at:
Nenhum comentário:
Postar um comentário