Karyotype variability in plant pathogenic fungi

2. Karyotype Variability in Plant Pathogenic Fungi
Seminar on
Chaithra,M.
PALM 7016
Department of Plant Pathology

3. Flow of Seminar
Introduction
Types of Variability in plant pathogenic fungi
Mechanism in a Chromosomal rearrangement
Consequences of Chromosomal rearrangement
Case studies
conclusion

4. • Karyotype is the number and appearance of chromosome in the nucleus
of an organism
• Variability: it is the property of an organism to change its characters from one
generation to theother
1Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB

5. 2Karyotype variability in plant pathogenic fungi, PALM7016 ;2018-2019,ACM, UASB
Importance of karyotype variability
 Many fungus are smaller in size and have a weak morphological differentiation of
chromosome among the different species
 To understand the various mechanism of creation of variability in fungi during its
life cycle
 To know the evolution of new virulence strains against resistance varieties
 To identify the arrangement of pathogenicity gene on the chromosome

6.  Recombination
 Heterokaryosis
 Parasexuality
 Mutation
 Cytoplasmic adaptation
3Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Variability in fungi

7. 4Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Variable karyotype
Chromosome
number
polymorphism(CNP)
Chromosome
length
polymorphism(CLP)
Wittenberg etal.,2009

8. Chromosome-numberpolymorphisms (CNPs)
Wittenberg etal.,2009
Nondisjunction:
“It is the failure of homologous chromosomes or sister chromatids to
separate properly during cell division”.
Ex: Mycosphaerella graminicola
5Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB

9.  Deletion
 Duplication
 Translocation
Ex: Magnaporthe grisea, Zymoseptoria tritici,
6Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Chromosome-length polymorphisms(CLPs)
Wittenberg etal.,2009

10. 7Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Chromosomal rearrangement
Fierro etal.,2017
“DNA shuffling throughout the genome and is a naturally occurring
heritable event in eukaryotic organisms”
 Source of genetic variation within and between individual species
 Role in the evolution of fungi
 Main cause of karyotype variability in populations

11. Mechanisms

12. Meiotic recombination
DNA repair Machinery
Parasexual recombination
Transposon – Associated Chromosomal rearrangement
Lateral DNA transfer
9Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB

13. 10Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
1. Meioticrecombination
Alexanderetal.,20091
Karyogamy
Failureto separate
(nondisjunction)

14. 11Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Meiotic transmission of unequal chromosome number in Mycosphaerella graminicola
Alexander et al., 2009
 Parental isolates : IPO323, IPO94269 and IPO95052
 Construct the linkage map of the both the hybrid and also construct bridge map by
using DArT and SSR marker
 Linkage group 8
 Genotyping of LG8 DArT markers
 PCR confirmation

15. 12Karyotype variability in plant pathogenic fungi, PALM7016,;2018-2019,ACM, UASB
Nondisjunctionresultsin lossofchromosomes
Graphicalgenotypingof LG8
Alexander et al., 2009

16. 13Karyotype variability in plant pathogenic fungi, PALM7016,; 2018-2019,ACM, UASB
Nondisjunctionresultsin disomy
Graphicalgenotypingof LG1 Alexander et al., 2009

17. 14Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
2. DNARepairMachinery
Double strand breaks
repairing pathways
Homologous
recombination repairing
pathway (HRR)
Non-homologous DNA
end joining
pathway(NHEJ)

18. 15Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Lieberetal.,2017
NonhomologousDNA
end-joining(NHEJ)
pathway
Spontaneousmutation
occursrandomlyat a
genomicposition
Ex: Cladiosporium fulvum, Zymoseptoria titici

19. 16Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
3. Parasexual recombination
 Parasexuality is a mechanism described in fungi that results in recombination in the absence of meiosis
 Guido Pontecorvo (1956): on Aspergillus nidulans (Emericella nidulans)
 Parasexual cycle is initiated by fusion of hyphae (anastomosis) during which nuclei and other
cytoplasmic components occupy the same cell (Heterokaryosis)
 Alternative source of recombination in imperfect fungi
eg: Colletotrichum acutatum, Cryphonectria parasitica : alternative source of genetic variability
 Also helps to transfer the pathogenicity chromosomes among asexual lineage of the fungi
Milgroom etal.,2017

20. 17Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Schoustraet al.,2007
Parasexual cycle in the filamentous fungus : Aspergillus nidulans
Anastomosis

21. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Schoustra et al., 2007
18
To study the specific advantages of haploidy or diploidy in the fungus
Aspergillus nidulans
 Evolving strains of Aspergillus nidulans
 Comparing the rate Relative fitness between
haploid and isogenic diploid strains based on
Mycelial Growth Rate

22. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
FitnessTrajectoriesof EvolvingStrains
(A) haploid strains (B)diploid strains
Eachline- one singleevolvingstrain
Haploidizeddiploidsare indicatedwith a red dashedline
Rate of adaptation of individual populations is estimated by the slope of the
fitness Schoustraetal.,2007
19

23. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Mean of all strains with respect to relative fitness
Fourreverted to haploidyin
the courseof the experiment
Diploidstrains
Haploidstrains
Mean rate of adaptation is given by the slope
Schoustraetal.,2007
20

24. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
4. Transposon –associated and repetitive element-associated CRs
 Transposable elements (mobile genetic elements)
 Tandem repeats (minisatellites and microsatellites)
 Segmental duplication
 Psuedogenes
Eg: Cladosporium fulvum – 90%
Blumeria graminis – 90%
Zymoseptoria tritici – 17%
Phytophthora infestans – 29%
Dhillon etal.,2017
21

25. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Intra-chromosomal mitotic recombination between repeats
Mehrabi etal.,2017
22

26. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Length of the spacer
sequence between the
repeated elements
Rate of intra-chomosomal recombination depends on:
Length of
the repeats
Orientation of the
two homologous
region
Eg: Cladosporium fulvum and Dothistroma septosporum : Intra-chromosomal
rearrangements by excision of transposons from chromosomes followed by inappropriate
NHEJ repair, significantly decreases the synteny between closely related species
Ohm et al., 2012
23

27. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
5. LateralDNAtransfer(LDT)
Mehrabi etal.,2017
Cytoplasmicfusionof two
different fungalspecies
An entire chromosome of a donar
species is transferred to the nucleus
of another species and proliferates
throughmitosis
Stable integration of a small
DNA segment from a
chromosome of one individual
to another
B
A
C
23
Donar
chromosome
24

28. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Lateral DNA transfer = Horizontal gene and chromosome transfer
 Transfer of (DNA segment) Pathotoxin producing genes
eg: T- toxin producing gene in Cochliobolous heterostrophus
HC-toxin producing gene in Cochliobolous carbonum
ToxA toxin producing gene in Pyrenophora tritici –repentis
 Transfer of entire chromosomes which is mainly responsible for pathogenicity
eg: Alternaria alternata
Walton etal.,2017
25

29. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Akagi et al., 2009
Horizontal transfer of an entire pathogenicity chromosome among the
Alternaria alternata strains
26
 Alternaria alternata : Alternaria stem canker on tomato
 Host specific toxin : AAL toxin
 Pathogenicity genes : ALT genes, Polyketide synthetase genes.
 Improper horizontal chromosome transformation : loss of chromosome
 This loosed chromosome called as conditional dispensable chromosome.

30. 27Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Dispensable chromosome / accessory chromosome
 One of the type of special chromosomes
 Randolph : coined this extra chromosome known as B chromosome
 It was first observed as extra chromosome in the hemipteran insect.
 It is unnecessary for survival and reproduction of an organism but involved in
pathogenicity or virulence on a specific host plants during the infection process
 This extra chromosome are known as B chromosomes, supernumerary
chromosomes, accessary chromosomes, (conditionally) dispensable chromosomes or
lineage specific chromosome or pathogenicity chromosome
Covert al.,2017

31. 28Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
 Alternaria alternata : Alternaria blotch in apple
 Isolates :O-210(original isolate) , O-210∆C(sub-culture strain)
 Identify the AMT and cyclic peptide synthetase gene in
original isolate and sub-culture strains of A. alternata by using
(RT)- PCR and leaf necrosis bioassay method
The effect of loss of dispensable chromosome on the
pathogenicity of Alternaria alternata
Johnson et al., 2001

32. 29Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
(RT)-PCR on Alternaria alternata strains
using AMT- specific primer to study
AMT gene expression
Leaf necrosis bioassay on susceptible apple laves using
culture filtrates of A. alternata strains to test for AM-
toxic production
Johnson et al., 2001

33. 30Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Colony morphology of A. alternata O-210
and 0-210∆c.
Pulsed –field gel electrophoresis of A.
alternata O-210 and O-210∆c
Johnson et al., 2001

34. 31Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
species Number
of DCs
Size range of
DCs
Functiona
l gene(s)
Biological significance
Nectria haematococca MPVI 3 0.45 to 1.6Mb PEP1.PEP2,
PEP5,PDA1
and PDA4
virulence on pea
Alternaria alternata 1 <2Mb Tox genes Pathogenicity on certain hosts
Fusarium oxysporum f. sp. Lycopersici 6 <3.5Mb Six genes Virulence on tomato
Colletotrichum gloeosporioides 1 2Mb Cyclic
homology
Unknown
Cochliobolus heterostrophus 1 1.2Mb Tox1 genes Virulence on maize
Cochliobolus carbonum 1 2.2 or 3.5Mb TOX2 genes Virulence on maize
Leptosphaeria maculans 1 0.73Mb AvrLm11 Virulence on oilseed rape
Zymoseptoria tritici 8 0.39 to 0.77Mb Unknown Quantitative virulence on
wheat
Magnoporthe oryzae 1 1.2Mb AVR-pita Virulence on rice
Gibberella fujikuroi MP A 1 0.7Mb unknown unknown
Known dispensable chromosomes (DCs) in filamentous fungi
Mehrabi etal.,2017

35. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Transformation-mediated loss of the 1Mb CDC of the A. alternata tomato pathotype
Leaf necrosis bioassay
for AAL toxin
production by the wild
type and mutant strains
Electrophoretic
karyotypes of
the CDC
deficient
mutant (9-1) &
wild type(As-
27) strain of
the A. alternata
Pathogenisity
test of the wild-
type and mutant
strains
Akagi et al., 2009
32

36. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Production of AAL and AF toxins and pathogenicity of the fusion strain EST6
Leaves of tomato and strawberry cultivar were
wounded Slightly, treated with culture filtrates of
the parent and fusion strain
Leaves were inoculated with mycelial pieces of the
strains incubate in a moist chamber at 250c for 3
days
Akagi et al.,2009
33

37. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Electrophoretic karyotypes of parental and hybrid stains of A. alternata
34
Akagi et al.,2009

38. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Menardo et al., 2016
Hybridof the two Powderymildewsspecializedontwo different hosts (Wheat andRye)caninfect
the hybridplant speciesoriginatingfrom those two hosts(Triticale)
Wheat Rye Triticale
35
hybridization

39. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Wheat powdery mildew is caused by Blumria graminis f. sp. tritici
Rye-B. graminis f. sp. secalis
Triticale -B. g. f. sp. triticale
Triticale was initially resistant to powdery mildew; however, this pathogen
was first observed on triticale in 2001 and has since become a major disease in
Europe
36

40. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
 The B.g. triticale genome consisted of genotype B. g. secalis
alternating with segments with a B. g. tritici genotype
 In B. g. triticale, between 11.9 and 21.4% of the polymorphic
sites represented the B. g. secalis genotype
 In contrast, over 80% of these genomes had the B. g. tritici
genotype
 Thus, they conclude that B. g. triticale is a hybrid of B. g. tritici
and B. g. secalis.
Menardo et al.,2016
37

41. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Hostspecificityofpowderymildewformae speciales
B. graminis f. sp.secalisXB.graminis f. sp.tritici
Wheat RyeB. graminis f. sp.triticale
triticale +wheat Menardo etal.,2016
38

42. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Model for the evolution of specialized forms and host ranges in B. graminis
Menardo et al.,2016
39

43. Wheat stem rust
Wheat stem rust : Puccinia graminis f. sp. tritici
New race TTKSK : Ug99 – virulence on Sr13+17 gene
New variant of Ug99: TTTSK – virulence on Sr36 gene & Sr13+17gene
Abrahim et al., 2018
Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB 40
Ethiopia

44. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Depotter et al., 2016
Hybridization mechanism
Genomic and transcriptomic consequences of hybridization
Pathogens on novel host through hybridization
Observed the naturally occurring interspecific hybrid pathogens and its importance on
genome evolution.
41

45. Karyotype variability in plant pathogenic fungi, PALM7016,;2018-2019,ACM, UASB
Examplesof naturally occurringinterspecifichybrid pathogens
Depotter etal.,2016
42

46. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Interspecific hybridization
Depotter et al., 2016
43

47. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Potential genomic and transcriptomic consequences of Allopolyploidy
44
Depotter et al., 2016

48. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Functional consequences of chromosomal rearrangements
Mehrabi etal.,2017
45
 Major evolutionary force for genetic diversity and adaptation to stressful
environments
 Host range alteration
 Disease outbreaks
 Emergence of virulent strains

49. Light microscopy
 Standard dyes such as giemsa or aceto-orceine
 Used only for fungal species capable of sexual reproductions
Germ tube burst method (GTBM)
 Better resolution
 Condensed mitotic metaphase chromosomes
 Magnification of chromosome size
 Used with conventional dyes in light and fluorescent microscopy
Pulsed-field gel electrophoresis (PFGE)
 Fungal Karyotyping
 Enable the visualization of small chromosomes and is independent of meiosis
 Used to estimate chromosome number and sizes of many fungal plant pathogen
Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Observation on karyotype variability
46

50. Karyotype variability in plant pathogenic fungi, PALM7016; 2018-2019,ACM, UASB
Adavanced molecular cytogenetic techniques…
47
Fluorescence in situ hybridization (FISH)
 Enabled detection of small deletion and duplication events that could not be
visualized by standard microscopy
Comparative genomic hybridization (CGH)
 Enables to compare two genomic DNA samples for gain or loss of entire
chromosomes or chromosomal segments
(Micro)array based CGH
 Where DNA microarrays are used instead of the traditional metaphase chromosome
preparation
 Detects very small alterations in chromosomes compare to FISH o traditional CGH