A talk at Beltwide Cotton Conferences 2021 (virtual).

1. Sameer Khanal1, Pawan Kumar2, Samantha Jo Wegener1, Richard Davis3, Robert L. Nichols4 and Peng Chee1
(1) University of Georgia, Tifton, GA, (2) USDA-ARS, Salinas, CA, (3) USDA-ARS, Tifton, GA, (4) Cotton Incorporated, Cary, NC
Transcriptome Analysis of Gossypium hirsutum Responses
to Meloidogyne incognita during Compatible and Incompatible
Interactions

2. Yield loss due to Meloidogyne spp. in the United States
 Yield loss increased from 1.0% in 1987 to
3.0% in 2017a
 Losses of more than 136.8 million kilograms
(2.78%) of cotton valued over $228.4
millionb
 Yield loss and cost of control due to plant
parasitic nematodes in Georgia, U.S. cotton
estimated at 132 million U.S. dollarsc
a Cotton Disease Loss Estimate Committee Report 2017
b Cotton Price Statistics Annual Report 2019
c Hajihassani et al. 2018

3. Recommended nematode management practices
 Cultural control
- Crop rotation and cover crops
 Biological control with nematopathogenic
bioagents
 Chemical control with nematicides
Host resistance is the best solution to root knot nematodes in cotton
Limiting factors:
• Extremely polyphagous with a broad host range
• Increase in cost of production
• Potent chemicals are often highly hazardous
• Unsustainable/economically infeasible in cotton

4. Host resistance against Southern root-knot nematode
Clevewilt 6
(moderately resistant)
Wild Mexican Jack Jones
(moderately resistant)
Auburn 623 RNR
(near immune)
M-lines (includes ‘M-120 RNR’, ‘M-315 RNR’)
(near immune; agronomically adapted)
Development of near-immune cotton
germplasm linesa
a Shephard et al. 1974; Shephard et al. 1989, 1996
b da Silva et al. 2019; Lopes et al. 2020; Jenkins et al. 1995; Wubben et al. 2020
c Shen et al. 2006; Shen et al. 2010; He et al. 2014; Kumar et al. 2016
Histopathological observationsb
two-stage post-penetration interference:
a.early (8–12 days) preventing juveniles from
developing functional feeding sites
b.late (25–30 days), impeding the development
of nematodes into eggs laying adult females
Molecular genetic studiesc
two major quantitative trait loci (QTLs):
a. qMi-CH11 curtails root gall production
b. qMi-CH14 limits nematode egg production

5. Fine mapping and candidate genes
a Shen et al. 2010
b Chen et al. 2020
c Kumar et al. 2019
Figure: Interval mapping (right) of root-knot nematode QTL in chromosome 11a, physical map
(left) of chromosome 11 in G. hirsutum genomeb and potential candidate genes showing
differential expression in a prior studyc.

6. Fine mapping and candidate genes
a Shen et al. 2010
b Chen et al. 2020
c Kumar et al. 2019
Figure: Interval mapping (right) of root-knot nematode QTL in chromosome 11a, physical map
(left) of chromosome 11 in G. hirsutum genomeb and potential candidate genes showing
differential expression in a prior studyc.
c

7. Fine mapping and candidate genes
a He et al. 2014
b Chen et al. 2020
c Kumar et al. 2019
Figure: Interval mapping (right) of root-knot nematode QTL in chromosome 14a, physical map
(left) of chromosome 14 in G. hirsutum genomeb and potential candidate genes showing
differential expression in a prior studyc.

8. Fine mapping and candidate genes
c
a He et al. 2014
b Chen et al. 2020
c Kumar et al. 2019
Figure: Interval mapping (right) of root-knot nematode QTL in chromosome 14a, physical map
(left) of chromosome 14 in G. hirsutum genomeb and potential candidate genes showing
differential expression in a prior studyc.

9. 0
50
100
150
200
250
300
4 DAI 8 DAI 12 DAI 16 DAI 20 DAI 25 DAI 30 DAI 40 DAI
NumberofM.incognita
Coker
M120
RKN Project (pilot): 4 libraries
Kumar et al. 2019

10. 0
50
100
150
200
250
300
4 DAI 8 DAI 12 DAI 16 DAI 20 DAI 25 DAI 30 DAI 40 DAI
NumberofM.incognita
Coker
M120
Current Project: 44 libraries
Khanal et al. (unpublished)
 2 genotypes (C201 and M120)
 2 treatments (RKN-treated vs. control)
 5 time points (4, 8, 12, 16 and 20 DAI)
 2 biological replications

11. Figure. Distribution of mapped and unmapped sequences corresponding to two different
genotypes (‘C’ C201 and ‘M’ M120), six different time points (‘0’, ‘4’, ‘8’, ‘12’, ‘16’ and ‘20’),
and two different treatments (‘Cont’ control and ‘RKN’ treated with RKN).
0
5
10
15
20
25
30
35
40
C00DAICont
C04DAICont
C04DAIRKN
C08DAICont
C08DAIRKN
C12DAICont
C12DAIRKN
C16DAICont
C16DAIRKN
C20DAICont
C20DAIRKN
M00DAICont
M04DAICont
M04DAIRKN
M08DAICont
M08DAIRKN
M12DAICont
M12DAIRKN
M16DAICont
M16DAIRKN
M20DAICont
M20DAIRKN
Numberofreads
(inmillions)
Mapped Reads Unmapped Reads
8.8 to 20.3 million paired-
end reads (average ~13
million reads)
Of 574.2 million total reads
obtained, 483.99 million
(84.28%) were mapped to
the reference 'TM-1'
genome (NAU-NBI_v1.1)a
a Zhang et al. 2015
Transcriptome sequencing and mapping

12. Differential expression of genes
Genotype Contrasts Downregulated Upregulated Total
C201 4DAI RKN vs. 4DAI Control 43 105 148
8DAI RKN vs. 8DAI Control 117 63 180
12DAI RKN vs. 12DAI Control 7 98 105
16DAI RKN vs. 16DAI Control 18 225 243
20DAI RKN vs. 20DAI Control 62 71 133
Total: 247 (30.5%) 562 (69.5%) 809
M120 4DAI RKN vs. 4DAI Control 4 66 70
8DAI RKN vs. 8DAI Control 312 409 721
12DAI RKN vs. 12DAI Control 95 82 177
16DAI RKN vs. 16DAI Control 179 314 493
120 20DAI RKN vs. 20DAI Control 65 136 201
Total: 655 (39.4%) 1,007 (60.6%) 1,662
Grand Total 902 (36.5%) 1,569 (63.5%) 2,471
Figure. Number of DEGs during compatible and
incompatible interactions at a. 4, b. 8, c. 12, d. 16
and e. 20 DAI. ‘C’ and ‘M’ correspond to C201 and
M120. Numbers following ‘C’ and ‘M’ correspond to
different DAI. ‘Up’ and ‘Down’ correspond to up-
regulated and down-regulated.
Temporal variation in transcriptional
responses corroborate histopathological
observations
- Highest number of DEGs at 8 and 16 DAI

13. Differential expression of genes
Genotype Contrasts Downregulated Upregulated Total
C201 4DAI RKN vs. 4DAI Control 43 105 148
8DAI RKN vs. 8DAI Control 117 63 180
12DAI RKN vs. 12DAI Control 7 98 105
16DAI RKN vs. 16DAI Control 18 225 243
20DAI RKN vs. 20DAI Control 62 71 133
Total: 247 (30.5%) 562 (69.5%) 809
M120 4DAI RKN vs. 4DAI Control 4 66 70
8DAI RKN vs. 8DAI Control 312 409 721
12DAI RKN vs. 12DAI Control 95 82 177
16DAI RKN vs. 16DAI Control 179 314 493
120 20DAI RKN vs. 20DAI Control 65 136 201
Total: 655 (39.4%) 1,007 (60.6%) 1,662
Grand Total 902 (36.5%) 1,569 (63.5%) 2,471
Figure. Number of DEGs during compatible and
incompatible interactions at a. 4, b. 8, c. 12, d. 16
and e. 20 DAI. ‘C’ and ‘M’ correspond to C201 and
M120. Numbers following ‘C’ and ‘M’ correspond to
different DAI. ‘Up’ and ‘Down’ correspond to up-
regulated and down-regulated.
Incompatible interaction i.e., between root
knot and resistant cotton show twice as
many DEGs than compatible interaction

14. Enriched categories of genes
a. b.
Figure. Enriched categories of DEGs during compatible and incompatible interactions. ‘Red’ bars
correspond to up-regulated and ‘blue’ down-regulated categories of genes in a. C201 and b. M120.

15. Example: alpha-linolenic acid pathway, a precursor of JA
Figure. Expression
profiles of the genes
encoding enzymes in
alpha-linolenic acid
pathway during
compatible and
incompatible
interactions between G.
hirsutum and M.
incognita at five
different timepoints (a)
and during incompatible
interaction at 8 DAI (b).

16. Early induction, greater diversity, and higher degree of
up-regulation of defense related genes: hallmarks of
incompatible interactions
Figure. Expression profiles of genes encoding enzymes involved in phenylpropanoid biosynthesis
pathway. DEGs at 8 DAI during incompatible interaction.

17. Example: transcription factors
Figure. Distribution of G.
hirsutum transcription factors
(TF) families during RKN
infection and development.
Down-regulated (a and c) and
up-regulated (b and d) TF
families in a compatible (a and
b) and an incompatible (c and
d) interactions. Corresponding
number of TF genes are
provided following TF family
identifiers (after comma).

18. General down-regulation of defense-related genes:
hallmarks of compatible interactions
For example:
 Universal stress and heat shock proteins, and genes encoding pattern recognition
receptors (PRRs) and Ca2+ signaling associated genes were generally down-
regulated during early infection (4 and 8 DAI)
 Defense pathways need to be repressed for a compatible interaction and nematodes
suppress key genes involved in ROS-mediated defense signaling during early stages
of infection
 Number of down-regulated genes was higher (117) than up-regulated (63) at 8 DAI
during compatible interaction.

19. DEGs at QTL regions as key RKN-
resistance candidates
Chr.a
Gene id Geneb
Description Genoc
DAI log2FCd
A11 Gh_A11G2836 RPPL1
Putative disease resistance RPP13-like
protein 1
M120 8 2.02
Gh_A11G2856 XYL1 Alpha-xylosidase 1 M120 8 -1.17
Gh_A11G2865 IQM3 IQ domain-containing protein M120 16 -1.92
D02 Gh_D02G0169 na na M120 12 1.09
Gh_D02G0191 At1g14780 MACPF domain-containing protein M120 8 1.34
Gh_D02G0196 WAKL8 Wall-associated receptor kinase-like 8 M120 16 7.25
M120 20 -7.51
Gh_D02G0205 BHLH62 Transcription factor C201 20 -1.18
Gh_D02G0206 CYS2 Cysteine proteinase inhibitor 2 M120 20 -3.64
Gh_D02G0213 na Tetrahydrocannabinolic acid synthase C201 16 2.08
Gh_D02G0216 CBDAS2 Cannabidiolic acid synthase-like 1 M120 8 2.93
Gh_D02G0218 na na C201 16 6.22
Gh_D02G0220 na C201 20 -1.61
M120 20 -2.26
Gh_D02G0241 SBP2 Selenium-binding protein 2 M120 16 2.33
Gh_D02G0242 SBP2 Selenium-binding protein 2 M120 16 6.34
M120 20 4.62
Gh_D02G0257 RLP12 Receptor-like protein 12 C201 16 3.53
Gh_D02G0259 RLP12 Receptor-like protein 12 M120 8 1.95
M120 12 1.62
M120 16 2.10
Gh_D02G0289 na Cytochrome P450 CYP749A22 M120 8 1.21
Gh_D02G0291 APL Myb family transcription factor M120 8 1.34
Gh_D02G0311 na
Glutamine synthetase leaf isozyme,
chloroplastic
M120 8 -1.68
Gh_D02G0328 GSTU7 Glutathione S-transferase U7 M120 8 2.01
Gh_D02G0326 GSTU7 Glutathione S-transferase U7 M120 20 2.69
Gh_D02G0327 na na M120 20 9.27
Gh_D02G0329 na Probable glutathione S-transferase C201 16 -1.41
Xyloglucan endotransglucosylase/hydrolase 2
a
Chromosome, b
na not available, c
C201 and M120 represent compatible and incompatible interactions,
respectively, d
negative log2FC are down-regulated. DEGs significant at adjusted p-value of 0.05 and

20. Conclusion
Almost immunity levels of resistance to RKN in M-120 RNR appears to be due to a greater
magnitude of expression of basal defense genes as well as because of differential induction
of M-120 RNR-specific genes, both in and outside of the QTL regions, distributed across the
genome
Nematode responsive genes related to defense pathways were often repressed during
compatible interactions, while earlier induction, greater diversity, and higher degree of up-
regulation of those genes were archetypal of incompatible interactions

21. Acknowledgement
• Dr. Peng Wah Chee (Supervisor)
• Dr. Richard Davis (USDA)
• Dr. Pawan Kumar
• Mrs. Rippy Singh
• Dr. Mychele Da Silva
• Cotton Molecular Breeding Lab. (CMBL)
members
• Dr. Bob Nichols
paulchong.netFunding source: