Plant material. The wheat near isogenic lines (NILs), with resistant (R) and susceptible (S) QTL-Fhb1, were developed from Sumai-3/Thatcher41 (seeds provided by Dr. S. Fox, AAFC, Winnipeg). Both R-NIL and S-NIL were homozygous resistant for the other reported type II FHB resistance QTLs on chromosomes 5A (QTL-Fhb5) and 6B (QTL-Fhb2) from Sumai-3. The NIL resistant to fusarium head blight (FHB) had high type II or resistance to spread of pathogen through rachis from the inoculated spikelet to other spikelets in a spike.
The Russet Burbank (RB) potato is a processing variety and the tissue cultured plantlets were obtained from the tissue culture centre, New Brunswick, Canada. The RB potato is on public domain. Experimental research, including the collection of plant material, complies with relevant institutional, national, and international guidelines and legislation and appropriate permissions were obtained for the collection of plant material.
HRC gene expression, sequence, and phylogenetic tree in wheat and potato. The physical location of the wheat QTL-Fhb1 has been variedly mapped to a 2.2–2.8 Mb region on the contig ctg0954 (Genbank: FN564434)42. An in-silico analysis revealed a gene encoding putative Sarcoplasmic/endoplasmic histidine rich Ca2+ binding protein at QTL-Fhb1 (Supplementary Table S1).
The total RNA was extracted using a RNeasy Plant mini kit (Quiagen). 500 ng of total RNA from each sample was reverse transcribed to cDNA using iScript cDNA synthesis kit (BioRad, ON, Canada). Two microliters of 40x-diluted cDNA in 10 µl reaction volume was used in a quantitative PCR (q-PCR) reaction using iQ SYBR Green Supermix (BioRad) in an CFX384TM Real-Time System (BioRad, ON, Canada). In wheat, relative transcript abundance in each sample was calculated by plotting onto the standard curve and was normalized with the reference gene, Ta2291 (T. aestivum ADP-ribosylation factor). In potato, normalisation was performed with the reference gene, β-tubulin (Stβ-tubulin) and elongation factor-1α (StEF1α) 43. Relative transcript abundance between treatments was calculated 2–∆∆CT method44 and compared by one-way ANOVA using SigmaPlot 12.5.
The HRC gene was searched in the potato genome and RNA-seq of pathogen inoculated Russet Burbank45, which was later Sangar sequenced in RB potato (Supplementary Table S1). The StHRC gene in RB potato was knocked out (Sthrc) based on CRISPR-Cas9. Guide RNA for StHRC Russet Burbank was designed using CRISPR-P 2.046 and was cloned in pDIRECT21A47. The final CRISPR-Cas9 constructs were delivered to potato internodes using Agrobacterium and plants were regenerated from internodal callus selected on Hygromycin48. The CRISPR induced mutation of all the four alleles of this gene was confirmed, and the CG1 (Clonal generation1) plant with biallelic mutation was selected for plant propagation, tuber production and disease quantification. The non-silenced (StHRC) and silenced (Sthrc) plants were further multiplied by tissue culture, in magenta boxes, containing MS (Murashige and Skoog) media and used in this study48.
The phylogenetic analysis of the reported HRC protein sequences was performed in Geneious® prime using the Genius plugin PhyML49 and a Kimura 2-parameter substitution model with bootstrap value of 1000.
Endonucleases in potato and differential gene expressions. The endonuclease gene AtCAN2 sequence27 was used to identify the ortholog in potato. A phylogenetic tree and amino acid sequence analysis identified four Clades and the StCaN2 belonged to the Clade-I. The RB potato plants with StHRC and Sthrc were inoculated with P. infestans and A. solani and the differential gene expressions were conducted.
Disease severity and pathogen biomass assessment. A pair of wheat spikelets in the middle region of spikes of both the NILs were inoculated (at 50% anthesis) with F. graminearum (isolate 15–35, provided by Dr. S. Rioux, CEROM, QC, Canada) macroconidial suspension (105 spores ml− 1) and the disease severity was quantified as per cent of spikelets in a spike diseased (PSD), at 3-day intervals, over 15 dpi. From the PSD, the area under the disease progress curve (AUDPC) was calculated24.
The leaves of RB potato plants, with functional StHRC and silenced Sthrc genes, were inoculated with Phytophthora infestans and Alternaria solani (obtained from Dr. A. Dionne, MAPAQ, QC) spore suspensions, and covered with plastic bags for 48 h. The disease lesion diameter was measured at 7 dpi; the cork borer discs containing the diseased area were collected at 7 dpi, and the pathogen biomass was quantified based on qRT-PCR assay with pathogen specific primers 48,50. Young tubers were inoculated with Streptomyces scabiei (isolate EF-35, obtained from Dr. C. Beauleau, Sherbrooke University, QC, Canada). The 1cm diameter cork borer discs were collected at 7 dpi and the bacterial biomass was determined based on real-time PCR assay using pathogen specific primers 48,51.
DNA laddering in wheat NILs and Russet Burbank potato plants, with HRC and hrc genes, inoculated with pathogens. Three alternative pairs of spikelets of NILs, with TaHRC and Tahrc, were inoculated with F. graminiarum, and the rachis samples were collected at 7dpi. Genomic DNA (gDNA) was extracted from the rachis, following 7 dpi with F. graminiarum, of wheat NILs using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany). 25 µg of DNA from pathogen inoculated NIL-R and NIL-S, 1 µg each of 100 bp and 1 Kb (Invitrogen, Carlsbad, CA, USA) were resolved on 2% agarose gel made with TAE buffer and stained with ethidium bromide. Gel images were captured using Molecular Imager ChemiDoc XRS System (Bio-Rad Laboratories).
The leaves of RB potato plants, with StHRC and Sthrc, were inoculated with P. infestans and A. solani. At 7dpi, a 1cm diameter cork borer was used to cut the diseased areas, ground and the genomic DNA was extracted using DNeasy Plant Mini Kit (Qiagen GmbH, Hilden, Germany). Genomic DNA was used to perform Ligation-Mediated Polymerase Chain Reaction (LMPCR) and DNA laddering52.
TUNEL assay for AL-PCD. Protoplasts isolated from StHRC and sthrc plants. StHRC and inoculated with methanol (control) and Thaxtomin A (2uM) (abcam Inc). TUNEL assay was performed after 72 hrs of Thaxtomin A treatment, using the dead end colorimetric TUNEL system (Promega corporation). Total of 500 cells were counted (in three replications) to calculate percentage of cell death37.
Metabolic profiling to identify and quantify metabolites. Three alternate spikelets of NILs were inoculated with mock or F. graminearum spore suspension and the spikelets and rachis from inoculated regions were separately harvested. The inoculated spike area was collected at 3 and 7 dpi, spikelets and rachis were separated, and metabolites were extracted and analyzed using liquid chromatography high resolution mass spectrometry (LC-ESI-LTQ-Orbitrap, Thermo Fisher, Waltham, MA)53,54. From the peak area or the abundances of metabolites, the Constitutive Resistance (CR) and Induced Resistance (IR) metabolites were identified55.
Table 1. Metabolites detected, in spikelets and rachises of wheat NILs with resistant and susceptible QTL-Fhb1 alleles, at 3 (a) and 7 (b) days post-inoculation with F. graminearum, respectively (Supplementary Table S2).
a: Induced resistance (IR) metabolites detected in spikelets at 3dpi.
Sl No.
|
Observed Mass (Da)a
|
Putative names of metabolites
|
Fold changeb
|
A. Phenylpropanpoids: Free phenylpropanoids, lignans, phenolic glucosides and flavonoids
|
1
|
148.0525
|
trans-cinnamic acid
|
1.24*c
|
2
|
164.0473
|
4-coumaric acid
|
1.45***
|
3
|
206.0579
|
p-coumaroyldiketide
|
2.09*
|
4
|
210.0891
|
Sinapyl-alcohol
|
4.4*
|
5
|
238.084
|
Sinapic acid methyl ester
|
2.99*
|
6
|
250.1346
|
N-Caffeoylputrescine
|
1.11*
|
7
|
342.097
|
Caffeic acid 3-glucoside
|
1.64*
|
8
|
356.1104
|
1-O-Feruloyl-β-D-glucose
|
1.49*
|
9
|
358.1411
|
(+)-pinoresinol
|
1.24**
|
10
|
372.1203
|
(+)-sesamolinol
|
1.81***
|
11
|
414.1277
|
(-)-Podophyllotoxin
|
1.5*
|
12
|
520.1936
|
(-)-Pinoresinol glucoside
|
1.53**
|
13
|
540.1623
|
Cleistanthin A
|
2.34
|
14
|
540.1658
|
2''-o-p-Coumaroylaloesin
|
1.35
|
15
|
550.204
|
Medioresinol 4'-O-β-D-glucopyranoside
|
1.39
|
16
|
328.1306
|
2,3,4,6-Tetramethoxychalocone
|
1.54*
|
17
|
340.1306
|
6-Prenylnaringenic
|
1.3**
|
18
|
402.1128
|
8-p-Coumaroyl-3,4-dihydro-5,7-dihydroxy-4-phenylcoumarin
|
1.55*
|
19
|
422.1714
|
Lupinisoflavone G
|
1.14*
|
20
|
446.1239
|
Biochanin A- β -D-glucoside
|
1.34*
|
21
|
478.2769
|
3-Geranyl-4-2',4',6'-tetrahydroxy-5prenyldihydrochalcone
|
1.38***
|
22
|
500.13
|
Epigallocatechin 5,3',5'-trimethyl ether 3-O-gallate
|
1.2*
|
23
|
564.1068
|
Isorhamnetin 3-(6"-malonylglucoside)
|
2.53
|
24
|
580.2147
|
(+)-Syringaresinol O- β -D-glucoside
|
1.75
|
25
|
602.2352
|
5,4'-Dihydroxy-6-C-prenylflavanone 4'-xylosyl-(1->2)-rhamnoside
|
1.11
|
28
|
726.2354
|
Naringenin 7-O-(2'',6''-di-O-alpha-rhamnopyranosyl)-beta-glucopyranoside
|
1.43
|
29
|
742.2666
|
Acanthoside D or (-)-Syringaresinol di-beta-D-glucoside
|
1.29
|
30
|
800.2357
|
Tricin 7-rutinoside-4'-glucoside
|
1.56
|
31
|
862.2141
|
Cyanidin 3-[6-(6-p-hydroxybenzoylglucosyl)-2-xylosylgalactoside]
|
1.75
|
B. Lipids
|
32
|
136.0374
|
Threonate
|
1.24*
|
33
|
172.1464
|
Caprate
|
1.78*
|
35
|
438.261
|
(-)-Fusicoplagin A
|
2.74*
|
36
|
440.2766
|
PA(17:1(9Z)/0:0);1-(9Z-heptadecenoyl)-sn-glycero-3-phosphate
|
3.11*
|
39
|
600.2312
|
12S-acetoxy-punaglandin 2
|
1.56
|
C. Terpenoids and alkaloids
|
40
|
154.1359
|
(3S)-linalool
|
1.29*
|
41
|
240.0898
|
β-Carboline-1-propionic acid
|
1.3*
|
42
|
376.1361
|
Loganate
|
1.59*
|
43
|
376.1516
|
Euparotin
|
1.08***
|
44
|
376.1516
|
Ailanthone
|
1.54**
|
45
|
418.1623
|
Euparotin acetate
|
1.28**
|
46
|
522.2091
|
Isobrucein A
|
1.46*
|
b: Glycerophospholipids detected in rachis at 7dpi
Observed mass (Da)
|
Putative names of metabolites
|
RM/SM
|
RP/RM
|
SPd/ SM
|
432.2265
|
PA(18:3(6Z,9Z,12Z)/0:0)
|
1.01
|
0.94
|
0
|
453.2846
|
PE(16:0/0:0)
|
1.05
|
1.04
|
0
|
479.3001
|
PC(15:1(9Z)/0:0)
|
0.99
|
0.91
|
0
|
503.3
|
PE(20:3(8Z,11Z,14Z)/0:0)/LysoPE(0:0/20:3(11Z,14Z,17Z))
|
0.97
|
1.00
|
0
|
505.3154
|
PC(17:2(9Z,12Z)/0:0) or LysoPE(0:0/20:1(11Z))
|
1.07
|
0.93
|
0
|
507.3315
|
PC(17:1(10Z)/0:0)
|
1.01
|
0.90
|
0
|
508.3132
|
PG(19:0/0:0): 1-nonadecanoyl-glycero-3-phospho-(1'-sn-glycerol)
|
0.97
|
1.0
|
0
|
519.2594
|
PS(18:3(6Z,9Z,12Z)/0:0)
|
0.97
|
0.97
|
0
|
555.3524
|
PC(17:0/0:0);1-heptadecanoyl-sn-glycero-3-phosphocholine
|
0.96
|
1.12
|
0
|
559.2872
|
PS(19:1(9Z)/0:0): 1-(9Z-nonadecenoyl)-glycero-3-phosphoserine
|
1.06
|
0.96
|
0
|
567.3523
|
PS(21:0/0:0)
|
1.07
|
0.86
|
0
|
578.4295
|
PA(O-16:0/12:0)
|
1.06
|
0.98
|
0
|
581.368
|
PS(22:0/0:0)
|
1.07
|
0.89
|
0
|
638.414
|
PG(14:0/12:0)
|
0.85
|
1.17
|
0
|
662.4139
|
PG(14:1(9Z)/14:1(9Z))
|
0.92
|
1.12
|
0
|
a The metabolite mass was included only when the observed mass accurate mass error, AME was < 5ppm, which was calculated as [(Observed mass - Exact mass)/Exact mass] x 106. To the observed mass to which one 1H was added, where the mass of H = 1.007276, as the mass was detected based on negative ionization mode, using LC-HRMS. |
b Fold change in abundances of induced resistance (IR) metabolites in NIL-R, calculated as the abundance: IR = (RP/RM)/(SP/SM), where R is resistant NIL, S is susceptible NIL, M is mock inoculated, and P is pathogen inoculated. |
c Significance calculated based on student t-test P value: *** <0.001, ** <0.01, * <0.05, NS is Not-significant. |
d In Table 1b, SP/SM = 0, because these metabolites were absent in the SP, at 7dpi. |