Chapter 4 Phytotoxin

1. Chapter 4 Phytotoxin - Huang’s chapter 6- e book

2. Phytotoxin • Plant-pathogenic toxins • Produced by bacteria or fungi • The toxins may be glycosides, amino acid derivatives, peptides, terpenoids, sterols, pyridines, and quinones, etc.

3. Phytotoxin • Divided into two categories • Host-specific toxins • Host-nonspecific toxins

4. Toxins produced by bacteria • Most of the toxins are nonhost specific. • Chlorosis-inducing toxins • Tabtoxin – Pseudomonas syringae pv. tabaci (煙草野火病菌) (pv. stands for pathovar.) • Tabtoxinine-b-lactam • Inhibit glutamine synthetase which converts glutamic acid to glutamine. • Phaseolotoxin – Pseudomonas syringae pv. phaseolicola (葉燒病) • A tripeptide toxin • Rhizobitoxine – Rhizobium japonicum • Block methionine biosynthesis pathway (Continued)

5. Tabtoxin • Non-host-specific toxin that inhibit glutamine synthetase. • Pseudomonas syringae pv. tabaci vs. tobacco, bean, soybean (tobacco wildfire or halo blight disease, 煙草野火病) • Dipeptide, tabtoxinine-b-lactam moiety linked to either L-threonine or serine • Tabtoxin itself is biologically inactive, but is readily cleaved by amino-peptidase of bacteria or plants origin to yield the active moiety tabtoxinine-b-lactam (TbL). • Accumulation of the TbL in plants results in chlorotic halos surrounding the bacterial lesions. • TbL is not essential for pathogenicity. • The gene (tabA) responsible for tabtoxin production has been identified by Tn5 mutagenesis. It encode a enzyme involved in tabtoxin biosynthesis.

6. Soybean wildfire caused by Pseudomonas syringae pv. tabaci

7. Phaseolotoxin • Produced by Pseudomonas syringae pv. phaseolicola, which causes halo blight on bean (菜豆葉燒病) • Phaseolotoxin is an phosphosulfamylornithine-alanine-arginine tripeptide. • Soon after the toxin is excreted by the bacterium into the plant, plant enzymes cleave the tripeptide and release alanine, arginine, and phosphosulfamylornithine. Phosphosulfamylornithine is the biologically functional moiety of phaseolotoxin. • The toxin affects cells by binding to the active site of and inactivating the enzyme ornithine carbamoyltransferase, which normally converts ornithine to citrulline, a precursor of arginine. • Phaseolotoxin may act as a disease-resistance suppressor.

8. Halo blight (Pseudomonas phaseolicola) of beans

9. Toxins produced by bacteria • Wilt-inducing bacterial polysacchrides • Extracellular polysaccharidal slime (EPS) • Erwinia sp., Pseudomonas sp. and Xanthomonas sp. • Lipopolysaccharide (LPS) in Salmonella typhimurium and Shigella dysenteriae • Amylovorin – Erwinia amylovora (梨火傷病菌) • Glycopeptide toxins • Corynebacterium sp. , G (+) • Peptide toxins • Syringomycin – P. syringae • Hexapeptide

10. Bacterial wilt caused by Erwinia tracheiphila Sticky strand test on cut stems, with bacterial slime streaming from xylem tissues of cucurbit (葫蘆).

11. Relative viscosity and wilt-inducing effect of culture filtrate of P. solanacearum Culture filtrate relative viscosity wilting index Highly virulent 83 5 Weakly pathogenic 50 0 Avirulent 51 0

12. Fire blight (火傷病, Erwinia amylovora) of apple Devastation by fire blight of a 2-year-old high-density Gala apple orchard in Michigan after a violent storm. Other orchards in the path of the storm had similar losses.

13. Background: fire blight, caused by the bacteriumErwinia amylovora, are becoming increasingly common in Michigan’s high-value apple orchards. Orchardists in the west-central Michigan fruit belt experienced devastating losses after a severe rainstorm on 31 May 1998 that had winds estimated up to 140 miles per hour. Two weeks later, as predicted by a program for forecasting fire blight (MARYBLYT), fire blight symptoms began to show up on trees that had survived the storm. Then a hailstorm on 16 June further compounded the problem by spreading bacteria to fresh injuries. Young trees in high-density plantings are particularly vulnerable to infection. Cultivars exhibiting the greatest damage include Gingergold, Gala, and Jonathan. In early July, E. amylovora began to ooze from the rootstock of trees on Malling 9 and 26 rootstocks. The presence of E. amylovora in the ooze was confirmed by isolation of the bacteria and identification by PCR. In August, many apparently healthy trees will exhibit discoloration of the foliage and then collapse and die as the pathogen girdles the tree.

14. Toxins produced by fungi • Host-specific toxins • Host-nonspecific toxins

15. Host-specific toxins • Microbial metabolites have similar host specificity. e.g., a plant susceptible to a pathogen is sensitive to its toxin, whereas a plant resistant to a pathogen is insensitive to its toxin. • The virulence of the pathogenic strains is positively correlated to their capacity to produce the toxin. e.g., a highly virulent strains produce more toxin. • The toxin is able to produce symptom characteristic of the disease caused by the pathogen.

16. Host-specific toxins • Victorin (HV-toxin) – Cochliobolus victoriae (Helminthosporium victoriae) vs. oat (Leaf blotch, 燕麥葉枯病) • T-toxin (HMT toxin) - Cochliobolus heterostrophus (Helminthosporium maydis) race T vs. corn (southern corn leaf blight, 玉米葉枯病) • HS-toxin – Cochliobolus sacchari (Helminthosporium sacchari) vs. sugarcane (甘蔗眼點病) • HC-toxin – Cochliobolus carbonum (玉米葉斑病)(Helminthosporium carbonum) race 1 vs. corn • AK-toxin – Alternaria kikuchiana vs. pears (梨黑斑病) • AM-toxin – Alternaria mali vs. apple (蘋果黑點病) p. 54, Table 3.1 of e book

17. Leaf blotch (燕麥葉枯病) Helminthosporium victoriae (Perfect stage Cochliobolus victoriae)

18. Victorin (HV-toxin) • Host specific toxin • Victorin (HV-toxin) – Helminthosporium victoriae (Perfect stage Cochliobolus victoriae燕麥葉枯病) vs. oat variety Victoria • A cyclic pentapeptide • Victorin A, B, C, D (major), E • Victoria was introduced in 1945. Victoria and its derivatives contained the Vb gene for resistance to oat crown rust. • Oats contain Vb gene are susceptible to victorin. • Resistance to oat crown rust and susceptibility to the Helminthosporium victoriae are controlled by the same pair of alleles.

19. Victorin (HV-toxin) • Victorin is an unusual chlorinated peptide compound that target the Vb gene product on the host cells. • Vb gene encodes a victorin receptor (100kD membrane protein), which is a subunit of the glycine decarboxylase enzyme that catalyzes the conversion of two glycine molecules into one serine molecule. • This enzyme is essential for photorespiration and plant cells appear to go through an induced senescence and programmed cell death with cleavage of RUBISCO. (p. 55 of e book, continued)

20. Victorin (HV-toxin) • Increase permeability of the plasma membrane to electrolytes. • Dramatic changes in transmembrane potential, increasing respiration and ethylene production • Victorin is active against sensitive oats at 10pg/ml, but does not affect resistant oats at concentration one million fold (1mg/ml) higher.

21. T-toxin (HM-toxin) • In the 1960s, corns with the Texas male sterility cytoplasm trait (T-cms) was bred into much of the US maize for ease of hybrid production. • This trait confers male sterility on the maize. • In 1970, it was found that the corns with this trait was susceptible to Bipolaris maydis (Helminthosporium maydis) race T. • It was then found that the T-cms and T-urf13 is the same trait.

22. T-toxin (HM-toxin) • Host-specific toxin • Bipolaris maydis (Helminthosporium maydis) race T vs. corn (southern corn leaf blight,玉米葉枯病) • Race T appeared in the “corn belt” in 1969. By 1970, it had spread throughout the corn belt, attacking only corn that had the Texas male sterile cytoplasm. That is, corn with the Texas male sterile (Tms) cytoplasm is susceptible to the T-toxin, whereas corn with normal cytoplasm is resistant to the toxin. • The T race contains two genes, a polyketide synthase (PKS1) and a decarboxylase (DEC1) that are required for toxin biosynthesis. (p. 55 of e book, continued)

23. T-toxin (HM-toxin) • T-toxins are linear polyketols varying from C35 to C45 in length. • T-toxin binds to a 13 kD inner mitochondrial membrane protein (URF-13), the product of the T-urf13 gene, to create a pore on the membrane, cause leakage of small molecules, and subsequently inhibit ATP synthesis, resulting in cell death. • T-toxin does not seem to be necessary for pathogenicity of H. maydis race T, but it increase the virulence of the pathogen. See Figure 3.4 on p.56 of e book

24. Southern corn leaf blight

25. Texas male sterile (Tms) • T-urf13 is a gene in the Tms mitochondrial genome. • The gene encodes a membrane bound 13 kD polypeptide, URF13, which forms a pore in the membrane. • URF13 is associated with both disease-susceptibility and cytoplasmic male sterility. • HM-toxin binds to the URF13 protein in Tms mitochondria and inhibits ATP synthesis.

26. AM-toxin • Alternaira mali causes Alternaria blotch disease (蘋果黑點病) on apples. • Characterized by necrotic spots on leaves, shoots, and fruits of susceptible apples. • Cyclic tetradepsipeptide • 0.1 ~ 0.2ng/mL of AM-toxin are toxic to susceptible apples, whereas resistant apples are affected at a concentration of 104 ~ 105-fold higher.

27. Alternaria blotch (蘋果黑點病) on apples

28. HC-toxin (玉米葉斑病) • Produced by C. carbonum (northern leaf spot) • Does not directly cause plant cell death, instead it suppress plant defense responses. • The toxin is a cyclic tetrapeptide containing D-amino acids and inhibits histone deacetylase in the plant nucleus, causing hyperacylation of histone and changes in gene expression. (p. 55 of e book)

29. Nonhost-specific toxins • Toxic substances produced by phytopathogenic microorganisms can cause disease symptoms on the host plant as well as on other plants that are not normally attacked by the pathogen in nature. • The toxins do not have a role in the establishment of the pathogen in the host. It is able to induce same characteristics of the disease symptom. • Tentoxin – Alternaria tenuis • Fusicoccin – Fusicoccum amygdali vs. almond and peach Tabtoxin – Pseudomonas syringae pv. tabaci vs. tobacco, bean, soybean • Coronatine – Pseudomonas syringae pv. glycinea, maculicola, tomato vs. soybean, crucifer, and tomato • Phaseolotoxin – Pseudomonas syrinage pv. phaseolicola vs. bean and some legumes

30. Phytotoxin • Scheffer and Pringle (1967) group toxins as the pathogen-produced determinants of disease and group them into: • Primary determinants of pathogenicity • Secondary determinants of pathogenicity

31. Primary determinants of pathogenicity • Primary determinants are those essential for pathogenicity, including • Victorin (HV-toxin) • HC-toxin • AK-toxin • AM-toxin (They are all host-specific toxins.)

32. Secondary determinants of pathogenicity • Secondary determinants contribute to virulence of pathogen but do not control its pathogenecity, including • Tabtoxin • Strains of Pseudomonas syringae pv. tabaci produce tabtoxin cause the distinctive halo surrounding a necrotic lesion. Strains that fail to produce tabtoxin cause necrotic lesions without halos. • Coronatine • T-toxins (a host-specific toxin)

33. Coronatine • Coronatine is produced by a number of P. syringae pv. glycinea, maculicola, tomato vs. soybean, crucifer, and tomato • Various pathovars with Tn5 insertion mutation are still pathogenic. • Coronatine consists of two structural components, the polyketide coronafacic acid and the amino acid derivative coronamic acid. • Coronatine causes structure change of chloroplasts and chlorosis. • Coronatine may suppress the induction of defense-related genes by plants, allowing greater pathogen ingress and multiplication. • The biosynthetic genes are clustered in a 32 kb region of a 90 kb plasmid. (p.93 of e book)

34. Syringomycin • Produced by P. syringae pv. syringae • Cyclic lipodepsipeptides consisting of a b-hydroxy acid and nine amino acids • Four genes are involved in syringomycin production: syrA for regulation, syrB and syrC for biosynthesis, and syrD for transport.

35. Syringomycin • Elicits Ca+2-dependent callose synthesis in suspension-cultured cells. • Inhibit plasma membrane H+-ATPase activity in maize and storage tissue of sugar beet. • Increase respiration of succinate and NADH and the hydrolysis of ATP in isolated maize and pea mitochondria. • Causes K+ efflux, stomatal closure, and reduction in stomatal aperture of plants. => similar to ABA • Stimulates opening of Ca+2 channels of plasma membrane

36. Phytotoxins • Amino acid-derived and peptide phytotoxins • Derived from the acetate-mevalonate pathway • Derived from the acetate-polymavalonate route • Heterocycles

37. Amino acid-derived and peptide phytotoxins • Fusaric acid – Fusarium and Gibberella • Tenuazonic acid – Alternaria tenuis • Rhizobitoxine – Bradyrhizobium japonicum and Pseudomonas andropogonis • Coronatine – Pseudomonas syringae • Fumonisins – Fusarium sp. • AAL-toxin – Alternaria alternata f.sp. lycopersici • Triticones – Pyrenophora tritici-repentis • Cytochalasins and pyrichalasins – Phomopsis sp.

38. Peptide phytotoxins • Maculosin – Alternaria alternata • Sirodesmins – Sirodesmium diversum • Taxtomins – Streptomyces sabies • Tabtoxins – Pseudomonas syringae pv. tobaci • Phaseolotoxin – Pseudomonas syringae pv. phaseolicola • Tentoxin – Alternaria sp. • AM-toxin – Alternaria mali • HC-toxin – Helminthosporium carbonum • HV-toxin – Helminthosporium victoriae (p.55, 93 of e book)

39. Phytotoxins derived from the Acetate-mavalonate pathway • Phytotoxins with structures of terpenoid origin • Foeniculoxin – Phomopsis foeniculi • Eremophilanes – Macrophomina phaseolina • HS-toxin – Bipolaris sacchari (= Helminthosporium sacchari) • Fusicoccin – Fusarium amygdali • Colletotrichin – Colletotrichum sp.

40. Phytotoxins derived from the Acetate-polymavalonate pathway • T-toxin – Helminthosporium maydis • PM-toxin – Phyllosticta maydis • AK-toxin – Altenaria kikuchiana • Cercosporin – Cercospora sp.

41. Roles of phytotoxins in plant pathogenesis • As pathogenicity factors in plant-pathogen interactions • Victorin (HV-toxin) • HC-toxin • AK-toxin • AM-toxin • As virulence factors in plant-pathogen interactions • Tabtoxin • T-toxin • As disease-resistance suppressors • Phaseolotoxin • Coronatine

42. END