Verticillium dahliae isolates Vd1396-9 and Vs06-07, from Dr. Daayf’s lab collection, were grown on potato dextrose agar (PDA) media at 24°C for 2 weeks. V. dahliae Vd1396-9 and Vs06-07 have been characterized as highly and weakly aggressive isolates, respectively (Uppal et al., 2007; Alkher et al., 2009). The conidial spores were harvested by flooding culture plates with sterilized water, then the concentration was adjusted for each experiment.

The open reading frame (ORF) of ICSH1 was amplified from genomic DNA of isolate Vd1396-9 with specific primers flanked with restriction sites of EcoRI and BamHI (Table 1). The DNA fragment was cloned into the binary vector pDHT (Mullins et al., 2001) and mutagenized using the EZ::TN transposon system (Epicentre Technologies, Madison, WI, USA). Agrobacterium-mediated transformation of V. dahliae was conducted according to the method described by Dobinson et al. (2004). The transformants were selected in PDA media containing hygromycin B (50 μg/mL). PCR was performed to confirm gene replacement using ICSH1-Up-F upstream the target sequence and ICSH1-EcoRI-F (Table 1). The positive candidate transformants were selected and the expression of ICSH1 was confirmed by RT-PCR.

Verticillium dahliae was grown in CDB liquid media for 10 days, then mycelia were collected and ground in liquid nitrogen. The DNA extraction method followed the protocol of Al-Samarrai and Schmid (2000). Genomic DNA of V. dahliae (∼20 μg) was digested by restriction enzyme EcoRV overnight at 37°C, and the digested DNA was separated in a 0.8% agarose gel for 6 h at 70 V. The DNA was transferred to a nylon membrane following the protocol of Maruthachalam et al. (2011). Hybridization and detection were conducted with Amersham AlkPhos Direct Labeling and Detection Systems (GE Healthcare Life Sciences, Mississauga, ON, Canada). The signal was visualized on X-ray film (Kodak, Rochester, NY, USA).

Verticillium dahliae mutants (icsh1-2-3-1 and icsh1-2-12-1) and wild type were grown on PDA for 2 weeks, thereafter colony diameters were measured. Each isolate was replicated on 4 petri-plates. Five 1.2 cm diameter mycelial plugs were randomly chosen from 4 week-old V. dahliae PDA plates and placed into 40 mL sterilized water, vortexed well then conidia concentration was determined using a hemocytometer.

Potato cultivars Ranger Russet, a moderately resistant, and Kennebec, susceptible to Verticillium wilt, as well as the susceptible sunflower Hybrid IS8048 and the susceptible tomato variety Bonny Best were used in this study (Madhosingh, 1996; Alkher et al., 2009). Plants were grown in a soil mix containing sand, soil, and peat moss with 1:1:1 ratio in a growth room with a 16/8 h photoperiod and a 22/18°C temperature regimen.

Conidial suspensions of the V. dahliae highly aggressive isolate Vd1396-9, the weakly aggressive isolate Vs06-07 and the icsh1-2-12-1 mutant were adjusted to a concentration of 10⁸ conidia/mL, and 1 mL of each was placed into 100 mL Czapek-Dox Broth (CDB) liquid media (Difco Laboratories, Sparks, MD, USA), then incubated for 1 week at 23.5 ± 1°C. Potato tissue extracts were prepared according to the protocol of El-Bebany et al. (2011). Briefly, 5 g of root, stem, or leaf tissues from 3-week-old healthy Kennebec potato plants were ground into powder using liquid nitrogen with mortars and pestles. Twenty-five mL of sterilized distilled water was added to the powder, then placed on a shaker at 120 rpm (C2 Platform Shaker, Edison, NJ, USA) for 4 h. The mixtures were centrifuged at 2000 × g for 5 min, and the supernatant filtered through 0.45 μm syringe filters (Thermo Fisher Scientific, Wilmington, DE, USA). One milliliter of root, stem and leaf extracts were separately added to 1-week-old cultures in CDB media. The experiment was carried out in three replications for each isolate supplemented with different potato extracts. One milliliter of sterilized distilled water was added to the control. The mycelia were harvested a week after treatment, and immediately ground into a powder using liquid nitrogen with pre-chilled mortar and pestle. The mycelial powder was used for RNA extraction.

Healthy leaves were cut from 4-week-old Kennebec potato plants, and then petioles placed into 1 mL conidial suspensions of Vs06-07 or Vd1396-9, with a final concentration of 3 × 10⁷ conidia/mL to ensure quick infection. Sterilized water was used for control plants. A half milliliter of sterilized water was added to the detached leaves every second day. The detached leaves were kept at 24 ± 2°C, with a 16/8 h photoperiod day/night. Four to six leaves all from different potato plants were bulked as one replication, with three replications for each isolate for each time point (1 DAI, 3 DAI, 5 DAI, and 8 DAI). Gene expression was analyzed in all treatments. The leaf tissues were immediately frozen in liquid nitrogen, then ground to a fine powder and used for total RNA extraction.

Total RNA was extracted from potato tissues (100 mg) following the manufacturer’s protocol of the Omega Fungal RNA kit (Omega Bio-Tek, Inc., Norcross, GA, USA). The quantity and quality of the total RNA were analyzed using a NanoDrop 2000 (Thermo Fisher Scientific, Wilmington, DE, USA). The first DNA strand was synthesized from approximately one microgram of the total RNA using Superscript first strand synthesis kit (Life Technologies, Carlsbad, CA, USA).

Quantitative Real-Time RT-PCR was performed according to the protocol of SsoFast EvaGreen Super mix (Bio-Rad Lab, Philadelphia, PA, USA) using a CFX96 Thermal Cycler (Bio-Rad, Hercules, CA, USA). The expression level of the ICSH1 transcripts was assessed using specific primer sets (Table 1). The Histone H3 gene (VDAG_10035) was used as a reference gene. The data was analyzed using the 2^-ΔΔC_T method (Livak and Schmittgen, 2001).

The potato, sunflower and tomato were sown in small plastic trays containing LA4 soil mix (SunGro Horticulture, Agawam, MA 01001, USA) in a growth room for 3 weeks, then roots were washed, trimmed 1–2 cm from the tip and placed in a conidial suspension. The conidia of V. dahliae icsh1 mutants and wild type were harvested with sterile water and diluted to a concentration of 10⁶ conidia/mL. Sterile water was used as a control treatment. The inoculated plants were transferred to pots containing a pasteurized mixture of sand and soil (2:1). The plants were grown in the same growth room under temperature and photoperiod of 24/18 ± 2°C and 16/8 h day/night, respectively. The total AUDPC of percentage of infection, disease severity, and plant height were analyzed. The vascular discoloration of the stem cross-sections were rated in the last week according to Alkher et al. (2009). Biological replications for potato cultivars Kennebec and Ranger Russet, sunflower Hybrid IS8048, and tomato variety Bonny Best, were 12, 6, 4, and 6, respectively for each treatment. The experiment for potato cultivar Kennebec, sunflower Hybrid IS8048 and tomato variety Bonny Best were repeated with 3, 2, and 2 times, respectively with similar results.

Three-week-old plants of the potato cultivar Kennebec were inoculated using the root dipping method described above in one of the three conidial suspensions, including icsh1 mutant (icsh1-2-12-1), wild type strain Vd1396-9, or the sterilized water control. The leaves, stems, and roots were sampled at 4, 9, 14, 21, and 35 DAI with three replications. The same tissues were split and extracted for SA and JA analysis.

The extraction and HPLC-PDA-Fluorescence analysis for SA was performed according to El Hadrami et al. (2015). Briefly, 500 mg of potato tissues were ground into powder with liquid nitrogen, then suspended with 490 μl of 80% methanol and 10 μl of 12.5 μg/ml O-anisic acid (Sigma–Aldrich Canada Co.) as an internal standard (Meuwly and Métraux, 1993). The mixture was shaken overnight at 4°C and centrifuged at 5,300 rpm for 5 min., the supernatant was evaporated under N₂, then re-extracted by adding equal volumes of ethyl acetate twice. The acetate phases were combined into a new tube and dried under N₂. Finally, extracts were re-suspended in 500 μl pure methanol for free SA analysis. The aqueous phases from the previous step were used for bound SA extraction by hydrolyzing in an equal volume of 8N HCl at 100°C for 2 h followed by re-extraction in ethyl acetate. The samples were re-suspended in 500 μl pure methanol and run for SA on HPLC-PDA- Fluorescence (El Hadrami et al., 2015). Bound SA was determined by the amount of free SA released by hydrolysis.

The extraction and UPLC-MSMS analysis for JA was performed according to Henriquez et al. (2016). Briefly, 500 mg of potato tissues were ground into powder with liquid nitrogen, then suspended with 5 ml of extraction solvent (HCl:2-propanol: H2O in a ratio of 2:1:0.002) and 50 μl of 1 μg/ml ( ± )-9,10-dihydrojasmonic acid DHJA (OlChemIm Ltd. Czech Republic) as an internal standard. They were shaken for 30 min at 4°C and mixed with 7 ml of dichloromethane, shaken again for an additional 30 min followed by centrifugation at 5300 rpm for 5 min. The lower phase was transferred into a new tube, dried under N₂ on ice, then re-suspended in 500 μl 50% methanol.

Statistical analyses were performed using PROC MIXED with Statistical Analysis Software (SAS) (SAS Institute, Cary, NC, USA; release 9.1 for Windows). Data were checked for normality with PROC UNIVARIATE and outliers were removed based on residuals comparison to critical values for studentized residuals (Lund, 1975) and the Shapiro–Wilk test for normality. Gene expression data were normalized by log¹⁰ transformation. Mean values were separated using least squared means and letters assigned by the macro PDMIX800.sas (Saxton, 1998) with α = 0.05.

According to previous research, Vd1396-9 is a highly aggressive V. dahliae isolate on potato, while Vs06-07 is a weakly aggressive one (Uppal et al., 2007; Alkher et al., 2009). We tested the expression of ICSH1 in isolates with high and low levels of aggressiveness and under the elicitation of potato tissue extracts. The highly and the weekly aggressive isolates of V. dahliae were exposed to potato extracts from different plant parts. The expression of the ICSH1 increased in response to all extract types, but was significantly higher, in both isolates, in response to the root extract than to the leaf and stem extracts. There were no significant differences between responses of the highly and weekly aggressive isolates (Figure 1A).

The expression level of ICSH1 was firstly analyzed in the highly and weakly aggressive isolates during the infection of susceptible potato plants (cv. Kennebec), but due to the low biomass of the weakly aggressive isolateVs06-07, it was not detected by QRT-PCR in the plant tissues (Data not show). Therefore, the inoculation was tested with conidial suspensions on detached potato leaves to analyze the expression of ICSH1 at different time points after inoculation. The gene expression was analyzed for all the treatments, but there was no expression of ICSH1 on detached leaves without V. dahliae infection (Supplementary Figure S1). The expression level of ICSH1 transcripts in inoculated potato leaves followed the same trend with the two V. dahliae isolates tested. The expression started by increasing to a high level after 1 day and decreased thereafter. However, a significantly higher expression level was observed with the highly aggressive isolate Vd1396-9, as compared to the weekly aggressive one Vs06-07 (Figure 1B).

PAL and ICS are two plant pathways that are both involved in SA synthesis (Wildermuth et al., 2001; Pasqualini et al., 2003). In a previous study, PAL1 and PAL2 were shown to be up-regulated in potato in response to V. dahliae infection, indicating that PAL-mediated SA signaling pathway is involved in potato plant defense against V. dahliae (Derksen et al., 2013a). The expression of a key enzyme of the ICS pathway, Isochorismate synthase (StICS), was assessed during inoculation using the two isolates with contrasting levels of aggressiveness. The expression of isochorismate synthase (StICS) in potato under V. dahliae infection increased significantly 5 DAI, but with no significant difference between the tested isolates (Figure 1C).

To determine the function of ICSH1 in V. dahliae, Agrobacterium-mediated transformation was carried out to introduce the transposon insertion into V. dahliae Vd1396-9 and disrupt the ICSH1 as described by Dobinson et al. (2004). A DNA fragment containing the chloramphenicol resistance gene and the hygromycin phosphotransferase gene inserted at the 449th bp of ICSH1 ORF, was generated in the binary vector pDHt. Five positive icsh1 mutants were identified by PCR (Figure 2A). The integration of the transposon cassette was confirmed by southern blot (Figure 2B). On the RNA level, the depletion of icsh1 transcripts was confirmed by semi-quantitative PCR. The wild type and the ectopic control (ICSH1-ECT-2-11-3) (Random insertion in V. dahliae genome but without replacing the original ICSH1 ORF) showed normal levels of ICSH1 transcripts (Figure 2C). Out of the five identified mutants, two (icsh1-2-3-1 and icsh1-2-12-1) were selected randomly for the next steps of phenotype analysis.

The phenotype, growth rate, conidiation, microsclerotia formation of icsh1 mutants were tested on PDA plates, while the pathogenicity was tested on susceptible and moderately resistant potato cultivars, on sunflower and on tomato plants. The morphology, conidiation and microsclerotia formation were not affected in the tested V. dahliae icsh1-2-3-1 and icsh1-2-12-1 mutants (Figure 3). There was no significant difference among the icsh1 mutants and the wild type neither in growth rate nor in conidial formation. On the other hand, the total area under disease progress curve (AUDPC) of infection and disease severity showed significant reduction with V. dahliae mutants compared to the wild type in both susceptible and moderately resistant potato cultivars (Figures 4, 5). The plant height and vascular discoloration followed the same trend in both cultivars, with no significant difference between the mutants and the wild type. In addition to potato, the wild type and icsh1 mutants were tested on sunflower and tomato plants. In sunflower, both wild type and icsh1 mutant were able to induce disease, but there were no significant differences between wild type and mutants in the total AUDPC of either infection or disease severity (Supplementary Figure S2). In tomato plants, there were no significant differences observed in any of the tested parameters for both pathogen isolates (Supplementary Figure S3). In conclusion, the ICSH1 is important for virulence of V. dahliae on potato but not on sunflower and tomato.

To determine potential roles of other genes from the isochorismatase family in presence and absence of ICSH1, the expression of five genes from the isochorismatase family in V. dahliae (Accession # VDAG_06346, VDAG_03530, VDAG_06170, VDAG_06688, and VDAG_08870) was tested by QRT-PCR in a highly and a weakly aggressive isolates, and in the isch1 mutant (icsh1-2-12-1) under induction with potato extracts. The same volume of water was added in the medium for each strain as a control to potato extracts. All data was analyzed using the 2^-ΔΔC_T method (Livak and Schmittgen, 2001). In both strains, the expression level of VDAG_06688 and VDAG_08870 was too low to be detected. The expression of VDAG_03530 increased in both the highly and weekly aggressive isolates in response to all types of extracts, without significant differences between the isolates (Figure 6A). The expression of VDAG_06170 increased in both the highly and weekly aggressive isolates in response to all types of extracts (Figure 6B), with a higher level in the weakly aggressive isolate in response to potato leaf and root extracts. In response to potato leaf extracts, the expression of VDAG_06346 increased more in the weakly aggressive isolate than in the highly aggressive one but such increase was similar in the two isolates in response to both stem and root extracts (Figure 6C).

Comparison of the expression of the above-cited genes in the isch1 mutant and the wild type Vd1396-9 under treatments with different extracts revealed the expression levels of VDAG_03530 and VDAG_06170 to be significantly higher in the isch1 mutant than those in the wild type isolate Vd1396-9 (Figure 7 and Supplementary Figure S4). To make these comparisons more clear, the data were shown in terms of expression of these genes in icsh1 relative to the wild type strain under each treatment (Figure 7). Both stem and root extracts induced a higher expression of VDAG_03530 and VDAG_06170 in the mutant compared with the wild type (Figures 7A,B). Moreover, compared with the wild type with no treatment, the expression of VDAG_03530 in isch1 mutant was about eight-fold higher in response to water (control), stem extracts, and root extracts (Supplementary Figure S4A). Similarly, the expression of VDAG_06170 in isch1 mutant in response to water (control), stem extracts and root extracts was about 7-fold, 5-fold, and 4-fold respectively to that of the wild type with no treatment (Supplementary Figure S4B). Inversely, the expression of VDAG_06346 was higher in the wild type isolate compared to the icsh1 mutant in response to root extract (Supplementary Figure S4C).

In conclusion, the expression of VDAG_03530 and VDAG_06170 in the mutants compensate that of ICSH1 transcripts, but this compensation is not apparent in presence of potato leaf extracts (Figure 7 and Supplementary Figure S4).

To determine the role of ICSH1 in altering the accumulation of SA and possibly JA, the icsh1 mutant (icsh1-2-12-1) and wild type strain Vd1396-9 were selected for this experiment. The susceptible potato cultivar Kennebec was inoculated by root dip in a conidial suspension, while plant roots dipped in sterilized water were used as a control. The plant tissues were sampled at 4, 9, 14, 21, and 35 days after infection. Quantification of SA in different parts of potato plants after inoculation with V. dahliae wild type strain and the icsh1 mutant was done using HPLC-Fluorescence. SA levels were significantly higher in all infected plant parts with both wild type and the icsh1 mutant.

In the roots, the level of free SA in response to inoculation went higher at 14 DAI thereafter decreasing compared to the water control (Figure 8C). At early stages of leaf infection (9 DAI), bound SA accumulation was significantly higher in response to the icsh1 mutant, compared to the wild type, whereas the opposite happened in the roots (Figures 8D,F). In stems, there were no significant differences between the SA accumulation in response to V. dahliae inoculation and water control until 35 DAI (Figures 8B,E). In leaves, SA levels were significantly higher in response to the wild type, compared to the icsh1 mutant, starting from 21 DAI for the free SA and 35 DAI for the bound-SA (Figures 8A,D). JA was also quantified in leaf, stem and root tissues of cultivar Kennebec after inoculation with V. dahliae wild type and icsh1 mutant using UPLC-MSMS. There were no significant differences between plants inoculated with the wild type and the mutant in terms of JA induction in the stems and roots, which was less than in the wounded control (Figures 8H,I). In the leaves, however, both the mutant and wild type induced more JA than the control at 21 DAI. At 35 DAI JA levels in the wild type were significantly higher than the icsh1 mutant or water control (Figure 8G).

In conclusion, potato accumulated SA in the roots at early infection stages and in the stems in both early and later stages, while the accumulation of SA and JA occurred in the leaves at later stages in response to V. dahliae infection. The SA and JA accumulation followed different trends in the roots and stems at the early stages, and similar trends in the leaves at later stages. The icsh1 mutant induced a high level of bound-SA in the leaves only at 9 DAI, and thereafter decreased. Taken together, the results indicate that the wild type V. dahliae isolate induced more free-SA than the mutant in the leaves, and more bound-SA in the roots. However, in absolute value, the total SA induced by the mutant in the leaves, at 9 DAI, is higher than the free SA induced by the wild type in the roots, stems, and leaves, combined (Figure 8).