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General topic: how do pathogen/pest populations respond to deployment of host resistance?

General topic: how do pathogen/pest populations respond to deployment of host resistance?. Selective effects of qualitative resistance (R genes) Natural vs. human-created systems How does evolution to virulence affect pathogens – is there a cost to virulence?

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General topic: how do pathogen/pest populations respond to deployment of host resistance?

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  1. General topic: how do pathogen/pest populations respond to deployment of host resistance? • Selective effects of qualitative resistance (R genes) • Natural vs. human-created systems • How does evolution to virulence affect pathogens – is there a cost to virulence? • Can durability of R genes be predicted • Selective effects of quantitative (partial) resistance • Evolution of pathogen populations with host-selective toxins (HSTs) • What affects evolutionary potential of pathogen populations

  2. Terminology • Using Vanderplank’s definitions: • Virulence: specific disease-causing ability • Aggressiveness: non-specific disease-causing ability • Other definitions are used, especially in host-pathogen co-evolution (natural systems, human infectious diseases) • Virulence: damage a pathogen strain causes to a host

  3. Selective effects of R genes on pathogen populations • Natural, co-evolving systems vs. artificial, agricultural/horticultural systems • In co-evolving systems • “Arms race” model (discussed in Holub, 2001, “The arms race is ancient history in Arabidopsis, the wildflower,” Nat. Rev. Genet. 2:516-527.) • Transient polymorphism: continual selective turnover of alleles; “selective sweeps” where R genes driven to extinction

  4. Evolution of R genes in local populations: Transient polymorphism model of co-evolving host-pathogen system (Holub et al) • Resistance alleles would be relatively young, nearly identical in sequence to susceptible alleles

  5. In contrast: the “trench warfare” model of natural (co-evolutionary) systems: • Balanced or “dynamical” polymorphism • Epidemics alternate with periods of high host resistance and low pathogen population • Resistance and susceptibility alleles would be old, differ greatly in DNA sequence

  6. Trench warfare model – natural system • Around Rpm1 deletion site, divergence between R and S alleles indicates Rpm1 polymorphism arose 9.8 Million years ago – old!

  7. Corroborating evidence: Puccinia coronata on Avena sterilis (wild oat) in Israel, a stable coevolving pathosystem in the center of origin • Virulence/avirulence polymorphisms are highly diverse – • extreme genetic diversity in the host (many resistance types) • matched by broad range of virulence patterns in pathogen • Accumulation of many R and vir genes • Natural selection prevented from going to fixation of R genes and Vir genes – balancing selection • Prevalence of low-reaction resistance types over 17 years; Pc genes effective in the 1960s and 1970s are still effective against a large proportion of isolates 20-30 years later • “…An example of stability of wild pathosystems that consist of natural mixtures of resistance and virulence.” Leonard et al, 2004, Phytopathology 94:505-514

  8. More on evolution of resistance and virulence in natural systems: • Tellier and Brown, 2007, Polymorphism in multilocus host–parasite coevolutionary interactions, Genetics,177: 1777–1790. • Thrall and Burdon, 1997, Host-pathogen dynamics in a metapopulation context: the ecological and evolutionary consequences of being spatial, Journal of Ecology 85:743-753 • Thompson and Burdon, 1992, Gene-for-gene coevolution between plants and parasites, Nature 360:121-597

  9. Some conclusions from natural systems • There is substantial polymorphism at R and Avr loci both within populations and across spatially separated subpopulations • Some of these polymorphisms are ancient, indicating that selective forces are preserving them • Spatial dynamics are key (see Thrall and Burdon): • The stability of host-parasite interactions strongly affected by spatial substructuring • Patchiness promotes persistence of resistance/virulence polymorphisms • Extreme “booms” and “busts” appear to be a feature of human deployment of resistance

  10. Pathogen populations evolve differently in human-influenced systems • R-gene breakdowns in agricultural systems tend to be dramatic and relatively complete • In artificial systems, humans constrain host evolution, deploy R genes in vast swaths • Pathogens evolve virulence • Humans deploy new R genes • Etc.

  11. Comomnly deployed wheat powdery mildew (Pm) resistance genes in NC % isolates virulent

  12. Less commonly deployed Pm genes in NC % isolates virulent

  13. How does evolution to virulence affect pathogens? • Is there a fitness cost to having a virulence gene? • Vanderplank, 1963 & 1968: • Releasing a new R gene causes directional selection: virulent pathotypes increase in frequency • When R gene is defeated, withdrawing it from production leads to “stabilizing selection”: pathogen races with unnecessary virulence genes are eliminated

  14. Types of natural selection Step 1 Step 2

  15. What is pathogen fitness? • The combined ability of an organism to survive and reproduce • Quantifiable • Reproductive rate • Infection efficiency • Aggressiveness (amount of disease caused) • Disease severity • AUDPC • Frequency of a strain relative to other strains can be used as an estimator of fitness

  16. Estimating fitness Genotype A: 10 lesions/lesion/day • Fitness (W) is a relative parameter -- expressed relative to the most fit genotype (with fitness = 1.0) • Relative fitness of B = WB: 0.9 Genotype B: 9 lesions/lesion/day • Selection coefficients (s) are also compared to estimate changes in fitness of isolates over time; they measure the intensity of natural selection on a genotype (e.g., Zhan et al, 2002, J. Evol. Biol. 15:634-647) • Selection coefficient = the proportion by which the fitness of a genotype is less than that of the most fit • selection coefficient (s) of most fit (most frequent strain) is set to 0, so its W = 1. Relative fitness of other isolates is 1 – s.

  17. For example: (Zhan et al) • Co-inoculate multiple strains in equal proportions early in season, let them compete throughout season • Measure end-of-season frequencies • Most fit strain = highest-frequency strain • Selection coefficient of Gi (the ith genotype) = si = 1 - pit pj0 1/t pjt pi0 Where p = frequency, t = time, and 0 = inoculation time 0 ≤ s ≤ 1. • Bottom line: we infer that end-of-season frequency of a strain reflects relative fitness • Selection coefficient measures the intensity of selection on that genotype (larger s = higher negative selection against that genotype). Fitness = 1 – s.

  18. Is there a cost to virulence? • How dissociate effect of virulence gene from pathogen’s genetic background? • Near-isogenic lines (NILs) of pathogen (Leonard, 1977, Phytopathology 67:1273-1279; Klittich & Bronson,1986, Phytopathology 76:1294-1298) • Averaging over large isolate collection (Leonard, 1969, Phytopathology 59:1851-1857) • Crosses to dissociate virulence alleles from genetic background (Bronson & Ellingboe, 1986, Phytopathology 76:154-158) • Mutants (Prakash & Heather, 1986, Phytopathology 76:266-269) • Genetic transformation (Keller et al., 1990, Phytopathology 80:1166-1173) • Site-directed mutagenesis (Lindemann and Suslow, 1987, Phytopathology 77:882-886)

  19. Xi et al. , 2003, Mycol. Res. 107:1485-1492 Fig. 1. Frequency of pathotypes E97-2 and H97-2 of Rynchosporium. secalis co-inoculated over 4 cycles on two barley cultivars: (a) ‘Earl,’ susceptible to E97-2, and (b) ‘Harrington,’ susceptible to both. E97-2 had complex virulence (to Earl, Harrington, and 5 more cvs). H97-2 had simple virulence (to Harrington and 1 other cv, not Earl).

  20. “E97-2, a pathotype with unnecessary virulence genes against a susceptible cultivar, had greater parasitic fitness compared with H97-2, a pathotype without unnecessary genes for virulence.” (Xi et al)

  21. Same pathosystem, different conclusion: • Among 8 R. secalis isolates, negative correlation between complexity on barley differentials in GH and competitive fitness in the field Abang et al, 2006, Differential selection on Rynchosporium secalis during parasitic and saprophytic phases in the barley scald disease cycle, Phytopathology 96:1214-1222. • “Our finding that virulence complexity was negatively correlated with fitness suggests that directional selection against unnecessary virulence operates in this pathosystem.”

  22. Aggressiveness scores negatively correlated with isolate complexity

  23. Opposing Vanderplank: little evidence of long-term cost to “unnecessary” virulences • Parlevliet, 1981, Stabilizing selection in crop pathosystems: an empty concept or a reality? Euphytica 30;259-269: • “stabilizing selection” occurs in two-step process • Avr -> vir temporarily decreases fitness • Selection for fitness modifiers that overcome loss of fitness • Evidence from antibiotic resistance: • In Bacillus subtilis, both fitness modifiers and alternative alleles amelioriate fitness losses (rifampicin resistance) (Cohan et al., 1994, Evolution, 48:81-95) • With time, antibiotic-resistant bacteria that initially are less fit in absence of antibiotic regain relative fitness via “compensatory mutations” (Morrell, 1997, Antibiotic resistance: road of no return, Science, 278:575-576)

  24. Contrary evidence from malaria parasite • In 1993, Malawi became the first country in Africa to replace chloroquine with the combination of sulfadoxine and pyrimethamine for the treatment of malaria. • At that time, the clinical efficacy of chloroquine was less than 50%. • Molecular marker for chloroquine-resistant Plasmodium subsequently declined in prevalence and was undetectable by 2001 • Chloroquine once again effective in Malawi. • How did the frequency of the drug-resistant allele change in Plasmodium?

  25. So: if you remove a defeated R gene from commercial production, will the corresponding virulence in the pathogen population decline in frequency? • One would expect this to happen IF virulence carries a fitness penalty • It’s assumed by some: • E.g.,“Deployment of disease resistance genes by plant transformation – a ‘mix and match’ approach,” Pink and Puddephat, 1999, Trends in Plant Science, 4:71-75

  26. “A great advantage of the strategy is that it does not depend upon a supply of new resistance genes….Existing resistance genes can be recycled. For example, if a gene, or gene combination, is withdrawn because the frequency of the matching virulence increases in the pathogen population it can be re-introduced when thefrequency of the matching virulence allele(s) reduces.” (Pink & Puddephat)

  27. Best guess: • In general, if R gene is removed from use, frequency of virulence may decrease, but will likely remain sufficient to “flare up” again if R gene is redeployed • But magnitude of fitness penalty and length of time needed to restore fitness via compensatory mutations probably varies from virulence mutation to virulence mutation • So R genes may vary in durability (compared “head to head,” i.e., deployed on equal acreage, etc.)

  28. Is there some way to predict durability of particular R genes? • Leach et al, 2001. Pathogen fitness penalty as a predictor of durability of disease resistance genes, Annu. Rev. Phytopathology 39:187-224. • Reviews evidence related to hypothesis that durability of a resistance gene is a function of the amount of fitness penalty imposed on pathogen.

  29. Differences in durability of single-gene mediated resistance • Some single R genes have proven durable – • Monogenic resistance to Fusarium oxysporum in crucifers has lasted 90 yrs • Lr34 resistance to wheat leaf rust (Puccinia triticina) has lasted over 30 yrs • Xa3, Xa4 resistance in rice to Xanthomonas oryzae durable 10-15 yrs before virulence emerged, still has some effect.

  30. Some avr genes contribute to fitness • Bacteria: Some avr genes control both virulence and avirulence (both elicitors of HR and agents of pathogenicity) • Xanthomonas spp. (bacterial pathogens of pepper, tomato, citrus, etc.) • avrBs2 -> protein that’s secreted into plant cells; avrBs3 family -> ability to multiply intercellularly; lesion length • Pseudomonas syringae pv. tomato • avrRpt2 -> blocks activation of defense responses

  31. Fungal genes with dual function in virulence and avirulence - necrotrophs • Cladosporium fulvum (tomato pathogen) • Elicitors Avr4, Avr9: Avr4 protects pathogen against chitinase (van den Burg et al, 2006, MPMI, 19:1420–1430) • Rynchosporium secalis (barley scald) • NIP1 = AvrRs1: kills host cells, releases nutrients • Magnaporthe grisea (rice blast) • AVR-Pita -> protease expressed after pathogen is inside plant; fitness function unclear

  32. Avr genes and fitness (summary) • Not all avirulence genes make a measurable contribution to fitness • Mutations in Xanthomonad genes avrXa10 and avrBs3 did not cause measurable loss in aggressiveness • Some do, and relative magnitude of contribution to fitness may vary, even within highly similar gene family • avrXa mutants to virulence exhibit different fitnesses (discussed later) • Avr genes may contribute to different fitness attributes • Intercellular multiplication • Exit of intercellular spaces to leaf surface, etc.

  33. So if avr genes affect fitness differentially, mutations in them should differentially affect fitness, and R genes corresponding to them should be differentially durable. • Fitness penalty due to loss of avr function may be compensated by functional redundancy • There may be many copies (e.g., PWL genes in M. grisea, or avrBs3 genes in xanthomonads) some functioning as fitness but not recognition factors, or vice versa • Some of structural requirements for fitness and avirulence may be different – it may be possible to lose avirulence function without losing virulence function • AvrPto single amino-acid mutants lost avr but not vir function • In some cases, amount of avirulence protein can be down-regulated to avoid induction while maintaining virulence function • Phytophthora parasitica produces v low levels of elicitin

  34. Can we PREDICT which R genes will be more/less durable? Example #1: • Bai et al knocked out individual avr genes in X. oryzae (2000, MPMI 13:1322-1329). • Assayed isolates for fitness (also growth on IR24 leaves, not shown) • Avr gene with biggest effect on fitness predicted to correspond to R gene with greatest durability: which one? avrXa7 Tested in field with NILs, natural inoculum. Over 3 yrs, durability of Xa7 > Xa10.

  35. Example #2: • Laugé et al: screen for elicitors (avr products) that are important virulence factors, then search for resistance (1998, PNAS 95:9014-9018) • Knew avr4 and avr9 could be knocked out without detectable fitness penalty (Cladosporium fulvum on tomato) • Knew ECP2 was important virulence factor because ECP2 knockouts only weakly pathogenic • Identified 21 lines with resistance beyond known R genes, challenged them with ECP2: 4 had HR • If virulence and avirulence do not involve separate domains in ECP2, new R gene Cf-ECP2 expected to be durable

  36. How do pathogen populations evolve in response to quantitative resistance? • Selection is operating on pathogen aggressiveness • non-specific pathogenic ability (usually polygenic) • the average amount of disease across several host cultivars with different degrees of partial resistance • Can be measured as AUDPC or disease at a particular timepoint • Model predicts increasing levels of quantitative host resistance select for increasing levels of damage by parasite, while qualitative host resistance does not have same effect (Gandon and Michalakis, 2000, Proc. R. Soc. Lond. B 267:985-990). • Not much empirical data!

  37. Do partially resistant hosts select for more aggressive pathogen populations? • Potato cyst nematodes (Globodera pallida) reared several generations on R potato cultivars had increased reproductive rate, those on S cultivars did not (Schouten and Beniers, 1997, Phytopathology, 87:862-867). • Greater multiplication by Lettuce mosaic virus in PR lettuce cultivars may have led to new, more aggressive viral pathotypes (Pink et al, 1992, Euphytica 63:169-174)

  38. Cowger and Mundt, 2002, Aggressiveness of Mycosphaerella graminicola isolates from susceptible and partially resistant wheat cultivars, Phytopathology 92:624-630 • Planted six wheat cultivars varying in level of partial resistance; natural epidemic developed • Epidemics are initiated by ascospore showers, then develop via rainsplash of conidia • Collected M. graminicola isolates early in growing season, and again late (after time for selection on each host) • Inoculated isolates on seedlings of same six cultivars in greenhouse (5-10 isolates bulked by cultivar of origin, field rep, and collection date) • Found no difference in aggressiveness of late-season isolates from MR vs. S cultivars. Then looked at difference between early and late collections:

  39. Mycosphaerella graminicola (Septoria leaf blotch) on wheat Cowger & Mundt, 2002

  40. Contrary evidence from Zhan et al: • Mean selection coefficients (s) for 6 isolates of M. graminicola lower on Madsen (MR) than on Stephens (S) • Pathogen populations collected from Madsen maintained highest genotype diversity, showed smallest change in genetic structure during one growing season • Supported hypothesis that PR hosts can retard rate of evolution in pathogen populations • However, Abang et al, 2006 Phytopathology 96:1214-1222 used similar method on Rhynchosporium secalis (barley scald) • Mean selection coefficients for 5 isolates were significantly higher on MR cultivar than on three S cultivars • Showed differential selection during parasitic and saprophytic phases – mutant pathotypes able to infect newly released resistant cultivars may grow in frequency more slowly than expected if they are good competitors during saprophytic phase.

  41. Mundt et al., 2002 Probably 1 major gene (Stb4?) No known major gene = start of commercial production

  42. Bottom line on PR • Majority of evidence is that moderate resistance selects for more aggressive pathogen strains than does susceptibility • But the breakdown of moderate resistance is likely slower than that of a single major gene

  43. Host-selective toxins (HSTs) • HSTs categorized as • Small proteins • Pyrenophora tritici-repentis PtrToxA (cause of tan spot in wheat) • Phaeosphaeria nodorum SnTox series • Cassiicolin (Corynespora cassiicola causes leaf fall on rubber) • Secondary metabolite clusters, e.g., AAL-toxins (Alternaria alternata on tomato) • Keep in mind: some fungal phytotoxins in both categories are not host-selective, rather they have broad activity: • Small proteins: • NIPs in Rynchosporium secalis (cause of barley scald) • Secondary metabolite clusters • Gibberellins and tricothecenes in Gibberella zeae = Fusarium graminearum (cause of Fusarium head blight or scab of small grains) • Aflatoxins (Aspergillus)

  44. First HSTs identified in 1930s-40s Many important fungal pathogens in the class Dothideomycetes produce host-specific toxins (at least 20 fungal spp.) Only active against specific host; most required for pathogenicity “Inverse gene-for-gene” system; HSTs are “agents of compatibility” (Walton, 196, Plant Cell 8:1723-33) How do fungal pathogen populations evolve in response to host resistance when HSTs are central to pathogenesis? Pyrenophora tritici-repentis causes red smudge on durum wheat kernels; purified Ptr necrosis toxin on wheat leaves No Disease Disease No Disease No Disease

  45. Not an HST, nor even a toxin! • Most are secondary metabolites, often clustered in fungal genome • Some are individual small proteins Wolpert et al, 2002, Host-selective toxins and avirulence determinants: What’s in a name? Annu. Rev. Phytopathol. 40:251-285

  46. Fig. 3. VIGS-mediated suppression of Hm1 results in barley susceptibility to CCR1… (b and c) Typical appearance of lesions caused by CCR1 on normal barley leaves (b) or leaves in which the Pds gene is suppressed via VIGS (c). (d–f ) CCR1-caused expanding lesions on barley leaves in which Hm1 was suppressed alone (d and e) or in combination with Pds ( f). (g) Resistant lesions of Hm1-suppressed barley to an HC toxin-deficient isolate of CCR1. HC-toxin detoxified directly by maize HM1, which encodes HC-toxin reductase (Sindhu et al, 2008, PNAS 105:1762-1767) • All grass species have Hm1 homologs • HC-toxin is not a HST • It’s the malfunction of Hm1 in maize that makes HC toxin host-specific • Why are dicots, which have no Hm1 homologs, resistant to HC toxin?

  47. Not an HST, nor even a toxin! • Most are secondary metabolites, often clustered in fungal genome • Some are individual small proteins Wolpert et al, 2002, Host-selective toxins and avirulence determinants: What’s in a name? Annu. Rev. Phytopathol. 40:251-285

  48. Evidence for very recent horizontal transfer of ToxA from P. nodorum to Ptr(Friesen et al, 2006, Nature Genet. 38:953-956) • Horizontal transfer: another force for increasing fungal pathogen diversity • Same ToxA gene present in Pyrenophora tritici-repentis (Ptr, cause of tan spot of wheat) and in Phaeosphaeria nodorum; high homology between ToxA genes • Tan spot wasn’t a problem in U.S. until first described in 1941 Tan spot ToxA P. nodorum P. tritici-repentis

  49. HSTs may be under diversifying selection Example: P. nodorum (anamorph = Stagonospora nodorum), cause of leaf and glume blotch of wheat (Septoria = Stagonospora, Leptosphaeria = Phaeosphaeria)

  50. Diversifying selection = disruptive selection; two ore more phenotypes favored simultaneously Types of natural selection Step 1 Step 2

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