Genome analysis of fungal pathogens

1. SUDHIR NAVATHE, Ph.D
(Mycology & Plant Pathology)
Scientist C, ICAR-AICWIP Wheat,
MACS-Agharkar Research Institute, Pune (India)
E-mail: sudhir.agro123@gmail.com / snavathe@aripune.org
Genome Analysis of Fungal Pathogens of
Cereal Crops
M o d er n M y c olog y L e c t u re S e r i es
Jo i ntly o r ga niz ed b y j o intly C B EC a n d M y c oa sia

2. Fungal genome resources used to populate the genome species list

3. Number of fungal
species from each
phylum and
subphylum with at
least one available
genome based on
various databases

4. Comparison between
the proportion of
known and sequenced
fungal species in the
subphyla of
Ascomycota (A) and
Basidiomycota (B). The
number of known
species were obtained
from Kirk et al. (2008).
Aylward et. 2017
IMA FUNGUS · 8(1): 1–15

5. Categories of significance identified in the 1090 sequenced fungal species

7. (A, B) Genome size
(C , D) number of
open reading frames
(ORFs) per million
base pairs (Mbp).
in the plant
pathogen genomes

8. ❖ Some
Major plant
pathogens
and
their hosts

9. SPECIES
(ISOLATE)
ISOL
ATE-
SPECI
FIC
HOST
SPECIE
S-
SPECIFI
C HOST
LIFE
STYLE
REPROD
UCTION
GENO
ME
SIZE
(MB)*
%
REPETITIVE
SEQUENCE*
GENOME FEATURES
LINKED TO
PATHOGENICITY‡
Bipolaris sorokiniana Wheat,
barley
Wheat, Barley Hemibiotr
op
Sexual and
asexual
35.7 ND Repeat-rich gene clusters that
encode effector candidates
Sporisorium
scitamineum (SscI8)
Sugar
cane
Sugar cane Biotroph Sexual 20 6.7 Repeat-rich gene clusters that
encode effector candidates
Ustilago maydis
(521) Maize Maize Biotroph Sexual 20 6.7 Repeat-rich gene clusters that
encode effector candidates
Microbotryum
lychnidis-dioicae
(p1A1 Lamole)
Red
campion
Red
campion
Biotroph Sexual 33 14 Repeat-rich gene clusters that
encode effector candidates
Melampsora larici-
populina (98AG31)
Poplar and
larch
Poplar and
larch
Biotroph Sexual
and
asexual
101 45 ND#
Puccinia graminis
f. sp. tritici
(CDL75-36–700-3,
race SCCL)
Wheat Wheat and
barley
Biotroph Sexual
and
asexual
89 45 Highly polymorphic effector
candidates
Zymoseptoria tritici
(IPO323) Wheat Wheat Hemi-
biotroph
Sexual
and
asexual
40 • 18.6 (genome
mean)
• 16.6 (core)
• 33.6 (accessory)
• Orphan regions are
enriched in in
planta-expressed genes
• Possible function of
accessory chromosomes in

10. SPECIES (ISOLATE) ISOLA
TE-
SPECIF
IC
HOST
SPECIES-
SPECIFIC
HOST
LIFESTYL
E
REPRODU
CTION
GENO
ME
SIZE
(MB)*
% REPETITIVE
SEQUENCE*
GENOME FEATURES LINKED
TO PATHOGENICITY‡
Blumeria graminis
f. sp. tritici (96224) Wheat Various Biotroph Sexual and
asexual
180 90 Presence and/or absence of
polymorphisms of candidate
effectors
Blumeria graminis f.
sp. hordei (DH14)
Barley Various Biotroph Sexual and
asexual
120 64 Repeat-rich accessory regions that
encode infection-specific
transcribed genes
Magnaporthe oryzae
(70–15) Rice Various crops
and wild
grasses
Hemi-
biotroph
Sexual and
asexual
41 10 Highly polymorphic effector
candidates and translocations of
effector genes
Fusarium graminearum
(PH-1)
Wheat Wheat and
barley
Hemi-
biotroph
Sexual
and
asexual
36 <3 Enrichment of in planta-
expressed and
species-specific genes in regions
of high SNP# density

11. Tools available for the analysis of fungal pathogen genomes

12. Major types of comparative genomics analyses used to identify unique characteristics of pathogen lineages

13. • The genome architecture of fungal plant pathogens is highly diverse.
• Genomes vary in total size, repeat content and gene number.
• The genome organization can vary from few chromosomes to many, and can include accessory chromosomes and particular
genome compartments.

14. Characteristics of Genome
Compartments
Specific characteristics that distinguish genome compartments from the core genome include lower gene density,
higher transposable element content, higher rates of evolution and distinct epigenetic marks;
LS, lineage-specific; RIP, repeat-induced point; TE, transposable element; V. dahliae, Verticillium dahliae.
Schotanus, K. et al 2015, Epigenetics Chromatin 8,

15. Fungal whole genome sequencing
workflow

16. 1.DNA Purity: OD260/280 =1.8 ~2.0 without
degradation or RNA contamination
2.Illumina platform: DNA/cDNA amount ≥ 2 μg,
concentration ≥ 20 ng/μL
3.PacBio platform: DNA amount ≥ 10 μg,
concentration ≥ 80 ng/μL

17. (A)General
workflow for
NGS.
(B)Bioinformatic
analysis
workflow for
metagenomic
data analysis.

18. Genome sequencing
• Sequencing platform:
PacBio/HiFi/Nanopore/Illumina
• Number of reads
• Read length (bp)
• Total read sequence (bp)
• Coverage (x)a
Assembly statistics
• Total genome size (bp)
• Number of contigs
• Number of scaffolds
• GC content
• N50 (Mbp)a
• L50b
• BUSCO completeness (%)
Annotation statistics
• Genes
• Secondary metabolism key
enzymes
• Carbohydrate-active enzymes
• Secreted proteins
• Candidate effectors
aLength of the smallest scaffold in an ordered set of scaffolds
corresponding to 50% of the genomes total size.
bSmallest number of scaffolds whose length equals 50% of the
genome total size.

19. Genomic Assembly
Genomic assembly and outcome evaluation
Coding/noncoding gene prediction
Repetitive sequence analysis
Genomic Structure
Analysis
Genomic island prediction
Prophage prediction
Detection of CRISPRs
LncRNA prediction
Functional Annotation NR, GO, COG, KEGG, SwissProt, Pfam, TCDB, SignalP, CAZy
Pathogenic Microbiology
Drug resistance gene database (ARDB)
Virulence factor database (VFDB)
The comprehensive antibiotic resistance database (CARD)
Comparative Genomic
Analysis
Synteny analysis
Analysis of gene family evolution
Phylogenetic analysis
ANI analysis

20. • Chromosome 7 of Zymoseptoria tritici has characteristics of both core and accessory chromosomes.
• The right arm is enriched with the heterochromatin-associated histone mark H3K27me3 and is poorly transcribed.
• A ribosomal DNA (rDNA) locus close to the start of the accessory region may be involved in the fusion of an accessory
chromosome to a core chromosome.
• A gene cluster that is potentially involved in virulence and the production of secondary metabolites is transcribed at the
right arm of the chromosome in the accessory-like region.
Chromosome 7 of Z. tritici has characteristics of core
and accessory chromosomes
Schotanus, K. et al 2015, Epigenetics Chromatin 8,

21. • Hybridization of two Blumeria graminis formae
speciales (f. sp.), B. graminis f. sp. tritici (blue)
and B. graminis f. sp. secalis (red), led to the
emergence of a new pathogen, B. graminis f. sp.
triticale, which infects the hybrid crop triticale;
a hybrid of wheat and rye.
• After the initial hybridization, the triticale-
infecting hybrid backcrossed with B. graminis f.
sp. tritici.
• This example illustrates an extraordinarily fast
adaptation to a new host that is mediated by a
combination of parental virulence factors
necessary for host infection.
Möller and Stukenbrock et al 2017,

22. Case Study
The discovery of the virulence gene ToxA in the
wheat and barley pathogen Bipolaris sorokiniana
McDonald et al 2018 (Australia)
Frisen et al 2019 (USA)
Navathe et al 2020 (India)
Wu et al 2020 (Mexico)
Black isolate White isolate
Mixed
isolate

23. McDonald et al 2018 Molecular Plant Pathology

24. (A) Total alignment of the 12-kb homologous region between the three species. This
view shows the differences in AT richness (green line); large increases are denoted by
red asterisks. The decay in identity near the edges of the region can be attributed to
repeat-induced polymorphism (RIP). All annotated genes are shown using the gene
accession number from P. nodorum (green bars).
(B) Differences in the promoter of ToxA as a result of the presence of small indels. In P.
tritici-repentis, there is a 238-bp insertion, which has no homology to any other organism.
In P. nodorum, a single 43-bp indel is present, which is a simple repeat of the same 43 bp
preceding the indel (blue bars). In B. sorokiniana, there is a 148-bp indel, which forms a
near-perfect DNA hairpin.

25. McDonald et al 2019, mBio
10.1128/mBio.01515-19
McDonald et al 2019 mBio

28. Global genomic analyses of wheat powdery mildew
reveal association of pathogen spread with
historical human migration and trade
Case Study
Sotiropoulos et al 2022
Nature communications
10.1038/s41467-022-31975-0
a global sample of 172 Blumeria graminis tritici (Bgt) genomes
analyses show that Bgt emerged in
• the Fertile Crescent during wheat domestication
• spread throughout Eurasia,
• colonization brought it to America, where it hybridized with
unknown grass mildew species.
• Recent trade brought USA strains to Japan, and European strains to
China.

29. Sotiropoulos et al 2022
Nature communications
10.1038/s41467-022-31975-0

30. Illumina paired-end sequence reads of 150 bp read length and an insert size of ca. 500 bp were used to generate 1 to
6 Gb sequence data per isolate using either HiSeq 4000 or NovaSeq 6000 technologies

33. https://doi.org/10.1038/s41467-023-36674-y

34. Draft and first Genomes of Plant Pathogenic Fungi
• Cercospora brassicicola
• C. Citrullina
• C. sesami
• Pseudocercospora cruenta
Upcoming
• Cercospora capsici
• Cercospora physalidis
• Cercospora abelmoschi
• Bipolaris sorokiniana

36. Recognised as reference
genome in database

37. A heat map of the
optimal pairwise
distance matrix showing
the significant
correlation among 21
available Cercospora
genomes

38. Summary of the distribution of orthologous clusters and protein data in the
compared Cercospora genomes

39. Venn diagram of the number
of shared and unique
proteins among Cercospora
species sequenced and
other related species

40. Where do you find the genomes of plant pathogenic
fungi?
Journal Publisher Article type Remarks
PhytoFrontiers The American
Phytopathological
Society (APS
Resource
Announcements
any species of plant pathogens, including those
with already published genomes, if the newly
reportedgenomic resource is of much better
quality. Subject to APC.
Genome Biology
andEvolution
Oxford University
Press
Genome Reports Only new genomes of organisms (not just
microbes) that are of interest to the broad
community of evolutionary biologists.
Subject to APC.
IMA Fungus BioMed Central IMA Genome Multi-authored articles that report the first
publicly available, high-quality
genomes of fungal species that are of interest
to the mycological community. NO APC
Microbiology
Resource
Announcements
American Society for
Microbiology
Genome Sequences Announcements of the availability of complete
genome sequences or draft
whole-genome sequences of any microbes to
the scientific community. Subject to APC.

41. GitHub Resources
• Keller-mark/awesome-biological-visualizations
https://github.com/keller-mark/awesome-biological-visualizations
• cmdcolin/awesome-genome-visualization
https://github.com/cmdcolin/awesome-genome-visualization
• PhyloT V2
https://phylot.biobyte.de/

42. Sequence analysis
❖ Links to Databases and web-based sequence analysis
http://www.soi.city.ac.uk/~drg/research/bioinformatics-list.html
❖ Protein and nucleic analysis
http://www.softberry.com/
❖ Secretion signal sequence and signal-peptide cleavage
analysis
http://www.cbs.dtu.dk/services/SignalP-2.0/

43. Sequences and sequence searches
✓ http://www.ncbi.nlm.nih.gov
✓ Blast GenBank nr or subsets of the database via the web. Also many other resources.
✓ Saccharomyces Genome Database (SGD). http://genome-www.stanford.edu/Saccharomyces/
✓ Yeast Proteome Database (YPD). Excellent reference source http://www.proteome.com/
✓ Candida albicans http://www-sequence.stanford.edu/group/candida/search.htm
✓ Neurospora Genome Project. EST sequences, RFLP mapping
http://biology.unm.edu/biology/ngp/home.html
Data and other links.
✓ Neurospora LG II/IV genome sequence with excellent annotation via PEDANT.
http://www.mips.biochem.mpg.de/proj/neurospora/
✓ Neurospora genome sequence http://www-genome.wi.mit.edu/annotation/fungi/neurospora/
✓ Neurospora, Aspergillus nidulans cosmids and ESTs http://www.genome.ou.edu/
✓ Schizosaccharomyces pombe genome sequence http://www.sanger.ac.uk/Projects/S_pombe/
✓ A. nidulans chromosome IV shotgun data and genome map http://aspergillus-genomics.org/
✓ Neurospora, A. nidulans, Nectria haematococca physical maps http://gene.genetics.uga.edu/
✓ Aspergillus flavus and Magnaporthe grisea ESTs and M. grisea
http://www.cals.ncsu.edu/fungal_genomics/

44. Li et al . Current Biology 2021 311653-1665.e5DOI:
(10.1016/j.cub.2021.01.074)
Copyright © 2021 Elsevier Inc. Terms and Conditions
Genome-
scale
Phylogeny
of fungi
Genome-scale phylogeny of the fungal kingdom based on 290 genes and 1,644 species

45. Our collaborators and Partners
1. Indian Council of Agricultural Research (ICAR)-Indian Institute of Wheat and Barley Research (IIWBR)
2. Consultative groups of International Research: CIMMYT, Mexico and ICARDA, Lebanon, BISA, India
Thank you for your kind attention!