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

Loss of the ability of Pseudomonas syringae pv. "phaseolicola" NPS3121 to elicit a hypersensitive response on tobacco and other nonhost plants was associated with loss of pathogenicity on the susceptible host bean. Eight independent, prototrophic transposon Tn5 insertion mutants which had lost the ability to elicit a hypersensitive response on tobacco plants were identified. Six of these mutants no longer produced disease lesions on primary leaves of the susceptible bean cultivar Red Kidney and failed to elicit a hypersensitive response on the resistant bean cultivar Red Mexican and on the nonhost plants tomato, cowpea, and soybean. The two remaining mutants had reduced pathogenicity on Red Kidney bean and elicited variable hypersensitive responses on the other plants tested. Southern blot analysis indicated that each mutant carried a single independent Tn5 insertion in one of three EcoRI fragments of about 17, 7, and 5 kilobases. Marker exchange mutagenesis further supported the conclusion that the pleiotropic mutant phenotype was not associated with multiple Tn5 insertions. A genomic library of the wild-type strain was constructed in the cosmid vector pLAFR3. A recombinant plasmid, designated pPL6, that carried P. syringae pv. "phaseolicola" genomic sequences was identified by colony hybridization. This plasmid restored the wild-type phenotype to all but one mutant, suggesting that genes affected by the insertions were clustered. Structural analysis of pPL6 and the wild-type genome indicated that the 17- and 5-kilobase EcoRI fragments were contiguous in the strain NPS3121 genome.

Gene cluster of Pseudomonas syringae pv. "phaseolicola" controls pathogenicity of bean plants and hypersensitivity of nonhost plants.

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.

Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves

In leaves of susceptible upland cotton (Gossypium hirsutum L.), Xanthomonas malvacearum (E. F. Sm.) Dows. produced expanding water-soaked areas, whereas in leaves of an immune cotton line, small clusters of dark brown host cells developed. The number of water-soaked areas or of brown cell clusters was proportional to the number of bacteria administered, when the initial bacterial population density was within the range 20 to 2500 bacteria per cm2 of leaf area. In paradermal sections of infected immune leaves, the brown host cells stained intensely with Azure B. Bacteria were found in the intercellular spaces adjacent to 64% of these darkly stained cell clusters and adjacent to only 7% of symptomless host cells. Compact masses of bacteria were observed on the surfaces of some darkly stained host cells. We conclude that in both susceptible and immune cotton leaves infection can be initiated by a single cell of X. malvacearum which grows into a colony. Bacterial growth was logarithmic in susceptible and immune leaves, but stopped at lower population densities in immune leaves. Any initial bacterial population density less than 850 cells per cm2 in immune leaves gave a growth yield of approximately 2000 per initial bacterium. This suggests that the immune leaf responds locally to each bacterial colony.

Single cell colonies of Xanthomonas malvacearum in susceptible and immune cotton leaves and the local resistant response to colonies in immune leaves

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

Paul E. Richardson

Papers

Adequacy of cellular phytoalexin concentrations in hypersensitively responding cotton leaves

Single cell colonies of Xanthomonas malvacearum in susceptible and immune cotton leaves and the local resistant response to colonies in immune 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

Sequential staining of callose by aniline blue and lacmoid for fluorescence and regular microscopy on a durable preparation of the same specimen.