Plants elaborate a vast array of natural products, many of which have evolved to confer selective advantage against microbial attack. Recent advances in molecular technology, aided by the enormous power of large-scale genomics initiatives, are leading to a more complete understanding of the enzymatic machinery that underlies the often complex pathways of plant natural product biosynthesis. Meanwhile, genetic and reverse genetic approaches are providing evidence for the importance of natural products in host defence. Metabolic engineering of natural product pathways is now a feasible strategy for enhancement of plant disease resistance.

Natural products and plant disease resistance

One of the best and longest-studied defense response of plants to infection is the induced accumulation of antimicrobial, low-molecular-weight secondary metabolites known as phytoalexins. Since the phytoalexin hypothesis was first proposed in 1940, a role for these compounds in defense has been revealed through several experimental approaches. Support has come, for example, through studies on the rate of phytoalexins in relation to cessation of pathogen development, quantification of phytoalexins at the infection site, and relationship of pathogen virulence to the phytoalexin tolerance. Evidence in support of phytoalexins in resistance as well some recent advances in phytoalexin biosynthesis are reviewed. Criteria for evaluating a role for phytoalexins in disease resistance are also discussed.

PHYTOALEXINS: What Have We Learned After 60 Years?

Plant antimicrobials are not used as systemic antibiotics at present. The main reason for this is their low level of activity, especially against gram-negative bacteria. The reported MIC is often in the range of 100 to 1,000 μg/ml, orders of magnitude higher than those of common broad-spectrum antibiotics from bacteria or fungi. Major plant pathogens belong to the gram-negative bacteria, which makes the low level of activity of plant antimicrobials against this group of microorganisms puzzling. Gram-negative bacteria have an effective permeability barrier, comprised of the outer membrane, which restricts the penetration of amphipathic compounds, and multidrug resistance pumps (MDRs), which extrude toxins across this barrier. It is possible that the apparent ineffectiveness of plant antimicrobials is largely due to the permeability barrier. We tested this hypothesis in the present study by applying a combination of MDR mutants and MDR inhibitors. A panel of plant antimicrobials was tested by using a set of bacteria representing the main groups of plant pathogens. The human pathogens Pseudomonas aeruginosa, Escherichia coli, and Salmonella enterica serovar Typhimurium were also tested. The results show that the activities of the majority of plant antimicrobials were considerably greater against the gram-positive bacteria Staphylococcus aureus and Bacillus megaterium and that disabling of the MDRs in gram-negative species leads to a striking increase in antimicrobial activity. Thus, the activity of rhein, the principal antimicrobial from rhubarb, was potentiated 100- to 2,000-fold (depending on the bacterial species) by disabling the MDRs. Comparable potentiation of activity was observed with plumbagin, resveratrol, gossypol, coumestrol, and berberine. Direct measurement of the uptake of berberine, a model plant antimicrobial, confirmed that disabling of the MDRs strongly increases the level of penetration of berberine into the cells of gram-negative bacteria. These results suggest that plants might have developed means of delivering their antimicrobials into bacterial cells. These findings also suggest that plant antimicrobials might be developed into effective, broad-spectrum antibiotics in combination with inhibitors of MDRs.

Multidrug Pump Inhibitors Uncover Remarkable Activity of Plant Antimicrobials

Summary The phytoalexin-deficient Arabidopsis mutant pad3-1, which is affected in the production of the indole-type phytoalexin camalexin, has previously been shown not to display altered susceptibility to either the bacterium Pseudomonas syringae (Glazebrook and Ausubel, 1994; Proc. Natl. Acad. Sci. USA, 91: 8955‐8959) or the biotrophic fungi Peronospora parasitica (Glazebrook et al., 1997; Genetics, 146: 381‐392) and Erysiphe orontii (Reuber et al., 1998; Plant J. 16: 473‐485). We now show that this mutant is markedly more susceptible than its wild-type parental line to infection by the necrotrophic fungus Alternaria brassicicola, but not to Botrytis cinerea. A strong camalexin response was elicited in wild-type plants inoculated with either Alternaria brassicicola or Botrytis cinerea, whereas no camalexin could be detected in pad3-1 challenged with these fungi. Hence, PAD3 appears to be a key determinant in resistance to at least A. brassicicola. The induction of salicylate-dependent and jasmonate/ethylene-dependent defense genes was not reduced in Alternaria-challenged pad3-1 plants compared to similarly treated wild-type plants. Camalexin production could not be triggered by exogenous application of either salicylate, ethylene or jasmonate and was not, or not strongly, reduced in mutants with defects in perception of these defenserelated signal molecules. Camalexin-production appears to be controlled by a pathway that exhibits little crosstalk with salicylate-, ethylene- and jasmonate-dependent signalling events.

https://onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-313X.1999.00513.x

Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola.

We are working to determine the role of the Arabidopsis phytoalexin, camalexin, in protecting the plant from pathogen attack by isolating phytoalexin-deficient (pad) mutants in the accession Columbia (Col-0) and examining their response to pathogens. Mutations in PAD1, PAD2, and PAD4 caused enhanced susceptibility to the bacterial pathogen Pseudomonas syringae pv. maculicola strain ES4326 (PsmES4326), while mutations in PAD3 or PAD5 did not. Camalexin was not detected in any of the double mutants pad1-1 pad2-1, pad1-1 pad3-1 or pad2-1 pad3-1. Growth of PsmES4326 in pad1-1 pad2-1 was greater than that in pad1-1 or pad2-1 plants, while growth in pad1-1 pad3-1 and pad2-1 pad3-1 plants was similar to that in pad1-1 and pad2-1 plants, respectively. The pad4-1 mutation caused reduced camalexin synthesis in response to PsmES4326 infection, but not in response to Cochliobolus carbonum infection, indicating that PAD4 has a regulatory function. PAD1, PAD2, PAD3 and PAD4 are all required for resistance to the eukaryotic biotroph Peronospora parasitica. The pad4-1 mutation caused the most dramatic change, exhibiting full susceptibility to four of six Col-incompatible parasite isolates. Interestingly, each combination of double mutants between pad1-1, pad2-1 and pad3-1 exhibited additive shifts to moderate or full susceptibility to most of the isolates.

https://www.genetics.org/content/genetics/146/1/381.full.pdf

Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance.

Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves

Development of fluorescent, hypersensitively necrotic cells containing phytoalexins adjacent to colonies of Xanthomonas campestris pv. Malvacearum in cotton leaves☆☆☆

Vernon E. Scholes

Papers

Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves

Development of fluorescent, hypersensitively necrotic cells containing phytoalexins adjacent to colonies of Xanthomonas campestris pv. Malvacearum in cotton leaves☆☆☆

A method for determining phytoalexin concentrations in fluorescent, hypersensitively necrotic cells in cotton leaves