Fungal & Oomycete Pathogenesis

Transcription

1 Fungal & Oomycete Pathogenesis - According to Agrios, >8,000 species of fungi can cause plant disease Latijnhouwers et al Trends in Microbiology 11:

2 Major differences between oomycete fungi and true fungi The spores of Phytophthora: weapons of the plant destroyer, Judelson & Blanco, Nature reviews of microbiology, 2005

3 Disease symptoms caused by phytopathogenic fungi with different lifestyles (a) Botrytis cinerea infecting tomato (b) Sclerotinia sclerotiorum infecting rapeseed (c) Uromyces viciae-fabae infecting bean (d) Blumeria graminis infecting barley (e) Ustilago maydis infecting maize (f) Cladosporium fulvum infecting tomato (g) Colletotrichum higginsianum infecting mustard spinach (h) Magnaporthe oryzae infecting rice

4 Plant colonization by fungi with different lifestyles Grow subcuticularly and kill epidermal cells by secreting toxic metabolites and proteins A complex series of developmental steps and eventually form a haustorial mother cell from which the haustorium, a balloon-shaped feeding structure, develops Develop bulged biotrophic invasive hyphae that later change into thin necrotrophic hyphae

5 Question: As a fungus, what might you need in order to infect a susceptible host?

6 General mechanisms involved in pathogenesis - Mechanical forces Formation of appressoria and penetration of the host cuticle and cell wall - Chemical weapons Enzymes: cutinases, pectinases, cellulases, hemicellulases, ligninases proteinases, amylases, lipases Toxins: non-host specific, host specific - Growth regulators auxins, gibberellins, cytokinins, ethylene, abscisic acid - Polysaccharides See chapter 3 of Plant Pathology by Agrios.

7 Examples of appressoria of fungi and oomycetes Features of appressoria of fungi: - Adhere to plant surface - Melanization - High turgor pressures - Form in response to appropriate signals Magnaporthe grisea, 3M glycerol, 8 MPa/1160 psi Latijnhouwers et al Trends in Microbiology 11:

8 The host haustorium interface Effectors Nutrient uptake Ann-Maree Catanzariti et al. FEMS Microbiol Lett 2007;269: Federation of European Microbiological Societies

9 Haustoria form contact between fungus and host Panstruga Curr. Op. Plant Biol. 6:

10 Fungal effectors Most fungal effectors are secreted, small, cysteine-rich proteins, have N- terminal signal peptide and a role in virulence. - Ace1 from M. grisea is the only known fungal effector protein that is not secreted by the fungus, but instead is suggested to be involved in the secondary metabolite biosynthesis. - Only two members of putatively cytoplasmic effectors which lack a signal sequence are Avra10 and Avrk1 of B. graminis f. sp. hordei. In resistant plants, effectors are directly or indirectly recognized by cognate resistance proteins that reside either inside the plant cell or on plasma membranes. Grouped into extracellular effectors that are secreted into the apoplast or xylem of their host plants and cytoplasmic effectors that are translocated into host cells. Fungal effector proteins, Ioannis & de Wit, Annual reviews 2009

11 Schematic representation of effector proteins Extracellular/Apoplastic effectors: A large number of effectors are located at the interface between pathogen and its host and fulfil a function on the outside of the host cell. Intracellular effectors: Several other effectors are able to translocate into host cells where they can, for example, interfere with defence responses of the host. Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges, Sonah et al FIPS, 2016

12 Examples of fungal and oomycete effectors Okmen Curr. Opin. Plant Biol. 20:19-25 Other reviews: Fungal effector proteins, Ioannis & de Wit, Annual reviews 2009 Filamentous plant pathogen in action, Giraldo & Valent, Nature reviews, 2013

13 Kamoun Curr. Opin. Plant Biol. 2007, 10:

14 Secretion of effectors into intercellular and intracellular spaces Blumeria graminis Cladosporium fulvum Melampsora lini Magnaporthe oryzae The role of effectors of biotrophic and hemibiotrophic fungi in infection, Koeck, Hardham & Dodds, Cell. Micro. 2011

15 Schmidt & Panstruga (2011) Curr. Opin. Plant Biol. 14: Analysis of fungal genomes - Secretome (Fungal Secretome Database, FSD - Effectoromics Effector-assisted breeding Effectors as Tools in Disease Resistance Breeding Against Biotrophic, Hemibiotrophic, and Necrotrophic Plant Pathogens (Vleeshouwers & Oliver, MPMI, 2014)

16 Flowchart of analytical tools to predict secretome and CSEPs in fungi Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges, Sonah et al FIPS, 2016

17 Computational Prediction of Effector Proteins in Fungi: Opportunities and Challenges, Sonah et al FIPS, 2016

18 N-terminal effector domains proposed to mediate host-cell entry How Do Filamentous Pathogens Deliver Effector Proteins into Plant Cells? Petre and Kamoun PLOS Biology, 2014

19 Mode and site of action of fungal effectors

20 Infection cycles of Ustilago maydis and Cladosporiuim fulvum Okmen Curr. Opin. Plant Biol. 20:19-25

21 Cladosporium fulvum Avr4 protects fungal cell walls against hydrolysis by plant chitinases accumulating during infection Cladosporium fulvum Avr4 binds to chitin van den Burg Mol. Plant Microbe Interact. 19:

22 Avr4 prevents chitin hydrolysis by tobacco chitinase van den Burg Mol. Plant Microbe Interact. 19:

23 Avr4 promotes growth of fungi in presence of chitinases van den Burg Mol. Plant Microbe Interact. 19:

24 Ustilago maydis Pep1 suppresses defense responses Hemetsberger PLoS Pathog. 8:e

25 Pep1 inhibits peroxidase activity and binds peroxidase 12 (POX12) Hemetsberger PLoS Pathog. 8:e

26 Silencing pox12 promotes infection by pep1 mutant Hemetsberger PLoS Pathog. 8:e

27 Layers of interaction between U. maydis and C. fulvum with hosts Okmen Curr. Opin. Plant Biol. 20:19-25

28 Plants present several barriers to pathogen infection: Cuticle Cell wall Suberin layers Seed coat from Agrios

29 How do fungal pathogens breach the physical barriers of plants? Direct penetration (physical and/or biochemical means) Go through natural openings Wounds Insect vector from Agrios, Plant Pathology, 3 rd ed.

30 Cell wall-degrading enzymes (CWDE) Fungi possess a diverse array of secreted enzymes to depolymerize the main structural polysaccharide components of the plant cell wall, i.e., cellulose, hemicellulose, and pectin. Recent advances in genomic and systems-level studies have begun to unravel this diversity and have pinpointed cell wall degrading enzyme (CWDE) families that are specifically present or enhanced in plantpathogenic fungi. These enzymes are particularly important for phytopathogenic fungi that do not have specialized penetration structures. Examples: Cellulose-degrading enzymes, hemicellulose-degrading enzymes, pectin-degrading enzymes. Plant cell wall-degrading enzymes and their secretion in plant-pathogenic fungi. Kubicek et al. Annu. Rev Phytopathol. 2014

31 The Oomycetes Oomycetes are another group of important plant pathogens. For a long time, oomycetes were classified as fungi because they have common characteristics. However, we now know they are genetically, morphologically and physiologically distinctive and belong to different taxonomic groups altogether. The most famous example of an oomycete disease is the Irish potato blight, caused by Phytophthora infestans, which changed the course of Irish/British history in the 19 th century. In fact, P. infestans still affects world potato growers today due to its rapid speed of adaptation to its host plant. Oomycetes can also infect other species; for example, the Saprolegnia genus infects fish.

32 Notable oomycete plant pathogens Phytophthora infestans, the potato late blight pathogen Plasmopara viticola, the cause of grapevine downy mildew Phytophthora cinnamomi, the cause of Phytophthora root rot of many plants Phytophthora ramorum, the cause of sudden oak death, Ramorum blight and shoot dieback Sclerophthora rayssiae var. zeae, the cause of brown stripe downy mildew of maize Pythium aphanidermatum and P. ultimum, causal agents of seed rot, seedling damping-off, and root rot /introoomycetes.aspx

33 Infection strategies and lifestyles of selected oomycetes Oomycete Interactions with Plants: Infection Strategies and Resistance Principles. Fawke et al. MMBR, 2015

34 Course of infection by Phytophthora infestans Stages of the spore cycles of Phytophthora infestans The spores of Phytophthora: weapons of the plant destroyer, Judelson & Blanco, Nature reviews of microbiology, 2005

35 Oomycete Interactions with Plants: Infection Strategies and Resistance Principles. Fawke et al. MMBR, 2015

36 Oomycete effectors Extracellular/Apoplastic effectors: A large number of effectors are located at the interface between pathogen and its host and fulfil a function on the outside of the host cell as in plant pathogenic oomycetes. Intracellular/Cytoplasmic effectors: Two important groups of translocated effectors are the RxLR-effectors and the crinklers found in abundance in many plant pathogenic oomycetes. Secretion, delivery and function of oomycetes effector proteins. Wawra et al. COIM 2012

37 Fungal and oomycete structures for effector secretion Petre PLoS Biol. 12:e

38 Several examples of apoplastic and cytoplasmic effectors Kamoun Ann. Rev. Phytopath. 44:41-60

39 Apoplastic effectors Protease inhibitors, cysteine-rich secreted proteins, and NPP1-family members Examples: Serine protease inhibitors-epi1 and EPI10 of P. infestans target the tomato protease P69B. Elicitors are molecules which stimulate a defense response in a host plant Most of them constitute pathogen-associated molecular patterns (PAMPs) because they are structurally conserved and thought to be indispensable components or products of a pathogen s life cycle or infection process. Elicitors are perceived by some plants as a microbial signature, likely through peripheral receptors, some of which require BAK1/SERK3 for their activity. Kamoun Ann. Rev. Phytopath. 44:41-60

40 Cytoplasmic effectors RXLRs P. sojae H. parasitica P. infestans First reported in 2005 the presence of a highly conserved amino acid motif, Arg-Xaa-Leu-Arg (RxLR), within avirulence proteins from different plant pathogenic oomycetes. This amino acid motif, often followed by an EER (Glu-Glu-Arg), is statistically enriched in the secretome of the Peronosporales, and positioned within the first 40 AA after the predicted signal peptide cleavage sites. These effectors are restricted to the Peronosporales clade, specifically Phytophthora and downy mildews. RXLR effector genes are not overrepresented in the genomes of Pythium ultimum and species outside the Peronosporales. Example: ~563 RXLR genes in the P. infestans genome. Kamoun Ann. Rev. Phytopath. 44:41-60

41 The RXLR motif present in a large class of oomycete effector proteins (Peronosporales Phytophthora & downy mildews primarily) Rehmany et al Plant Cell 17:

42 RXLR is a conserved host targeting signal Bhattacharjee et al PLoS Pathog 2(5): e50

43 RXLR is followed by sequences enriched for amino acids E, D, & R required for host targeting Bhattacharjee et al PLoS Pathog 2(5): e50

44 Distribution of RXLR genes in oomycete lineages Recent progress in RXLR effector research. Anderson et al MPMI 2015

45 Localization of two RXLR effectors Oomycetes, effectors and all that jazz. Bozkurt et al. COIPB, 2012

46 Assays for detecting pathogen-independent entry of effectors Kale and Tyler (2011) Cell. Microbiol. 13:

47 PI3P binding mediates entry of RXLR effectors Kale et al. (2010) Cell 142:

48 Model for entry of fungal and oomycete effectors Kale and Tyler (2011) Cell. Microbiol. 13:

49 Controversy over model for entry of fungal and oomycete effectors Ellis and Dodds (2011) Showdown at the RXLR motif: Serious differences of opinion in how effector proteins from filamentous eukaryotic pathogens enter plant cells. PNAS. 108: RXLR motif of AVR1b is not required for PIP binding Yaeno et al. (2011) PNAS 108: Wawra et al. (2012) Host-targeting protein 1 (SpHtp1) from the oomycete Saprolegnia parasitica translocates specifically into fish cells in a tyrosine-o-sulphate dependent manner PNAS. 109:

50 Petre PLoS Biol. 12:e xx xx

51 Oomycete RXLR effectors target diverse processes and cellular compartments to suppress immunity A, RXLR effectors enter host cells and localize to distinct compartments. B, Avrblb2 inhibits secretion of the C14 protease at the haustorial interface. C, IPI-O disrupts plasma membrane cell wall adhesions, in part through interaction with a host receptor-like kinase. D, HaRxL17 exerts its virulence activity at either the tonoplast, region surrounding the haustoria in infected cells, or both. E, Avr3b utilizes a Nudix motif to reduce production of reactive oxygen species. F, PexRD2 interacts with mitogen-activated protein kinase kinase kinase epsilon. G, HaRxL44 destabilizes the transcriptional regulator MED19a in the nucleus to activate jasmonic acid signaling and, thereby, suppress salicylic acid mediated immune responses. H, Phytophthora sojae PSR1 targets host RNA interference by interacting with an aspartate-glutamate-alanine-histidine box RNA helicase domain protein, which likely affects assembly of dicing complexes. Recent progress in RXLR effector research. Anderson et al MPMI 2015

52 Oomycete Avr genes Recent progress in RXLR effector research. Anderson et al MPMI 2015

53 Expression of AVR3a in P. capsici results in avirulence on R3a N. benthamiana leaves Sebastian Schornack et al. PNAS 2010;107: by National Academy of Sciences

54 Avr3a triggers HR in plants expressing R3a resistance gene Bos et al Plant J. 48:

55 AVR3a suppresses INF1-elicited cell death in N. benthamiana Avr3a is a cell death suppressor

56 Different amino acids affect R3a HR and suppression of cell death Bos et al MPMI. 22:269-81

57 CRINKLER (CRN) type effectors are common in oomycetes Leaf-crinkling and cell death phenotype observed during expression in planta. Crinklers (CRN) are cytoplasmic effectors first discovered in P. infestans. They are ubiquitous and have been detected in all examined plant pathogenic oomycetes such as P. sojae, P.ramorum, P. phaseoli, H. arabidopsidis, Bremia lactucae and Pythium ultimum. The CRN family is dramatically expanded in P. infestans (196 genes, 255 pseudogenes), whereas most other oomycetes have relatively fewer CRN genes, with P. ultimum having only 18. This group of effectors shares a similar modular structure with the RxLR-effectors exhibiting a highly conserved N-terminal Leu-Xaa-Leu-Phe-Leu-Ala-Lys (LxLFLAK) domain of approximately 50 amino acids. CRN proteins from P. sojae, P.ramorum and P. infestans do not have RXLR motifs but instead display the conserved motif LxLFLAK. CRN proteins in H. arabidopsidis do have RXLR motifs, and these overlap with LxLFLAK motif (eg. RKLRLFLAK). Oomycetes, effectors and all that jazz. Bozkurt et al. COIPB, 2012 Secretion, delivery and function of oomycetes effector proteins. Wawra et al. COIM 2012

58 The CRN protein family forms a class of modular proteins that are translocated inside host cells LXLFLAK translocation domain Sebastian Schornack et al. PNAS 2010;107: by National Academy of Sciences

59 CRN effector domains traffic to and function in the host nucleus Sebastian Schornack et al. PNAS 2010;107: by National Academy of Sciences

60 CHXC effector candidates represent a third delivery motif in oomycetes Found first in Albugo laibachii Kemen et al. (2011) PLoS Biology. 9:e

61 Summary Fungi and oomycetes have a variety of different lifestyles Common themes in fungal signal transduction controlling development and pathogenicity Oomycetes and fungi can form appressoria and haustoria Oomycetes and fungi can secrete a variety of effector proteins into intracellular and intercellular spaces Effectors perform functions in fungal walls, apoplast, and inside host cells RXLR & deer, LXLFLAK, and CHXC motifs mediate delivery into host cells Controversy over mechanisms of entry mediated by RXLR Question remains open on how effectors enter host cells.

62 RNAi- a molecular tool for breeding high-value crops Host induced gene silencing (HIGS) New wind in the sails: improving the agronomic value of crop plants through RNAi-mediated gene silencing. Koch & Kogel. PBJ 2014

63 sirna-mediated regulation in plant-pathogen interactions Small RNAs- the secret agents in the plant-pathogen interactions. Weiberg & Jin, COIPB, 2015

64 Questions for future research Filamentous plant pathogen in action, Giraldo & Valent, Nature reviews, 2013