Exogenous ACC impeded Vd991 infections of cotton plants
We first investigated whether ACC treatment affected the infection of V. dahliae on cotton plants. In no ACC treatment, the cotton seedlings inoculated with Vd991 displayed the typical disease symptoms, such as leaf yellowing, wilting and vascular browning (Fig. 1A and S1A). For cotton plants pretreated with ACC (25, 50 or 100 µM), the disease symptoms caused by Vd991 inoculation for 16 days was alleviated, for example, under the conditions of 100-µM ACC treatment, the disease index in the ACC-treated plants (28.69 ± 2.39%) was lower than that in the untreated plants (52.39 ± 3.29%) (Fig. 1C and S1B). Meantime, there was a lower fungal recovery from the stem sections collected from the inoculated plants (Fig. 1B), with the fungal biomasses in stems having decreased by 27% in the treated plants compared with in untreated plants (Fig. 1D and S1C). These data suggested that ACC was involved in the resistance response of upland cotton to V. dahliae infection, and the Vd991-associated degree of Verticillium wilt disease was negatively correlated with ACC concentrations.
To clarify whether ACC directly inhibited the reproduction of Vd991 stain, we checked the growth of mycelia and sclerotia on PDA medium exposed to ACC (0, 25, 50 or 100 µM). Results showed that the diameters of the growth of mycelia and sclerotia were no difference (Fig. S2), which suggest that ACC itself did not inhibit the fungal reproduction or growth. It is speculated that ACC may act as a regulator to enhance cotton host resistance to V. dahliae infection.
GhACS2/6-denpendent ACC accumulation improved cotton resistance to Vd991 infection under laboratory conditions
To understand the relationship between in vivo ACC production and V. dahliae infection, we investigated whether a Vd991 infection affected the expression of GhACS genes in RNA-seq database (Genome sequencing project accession: SRP118279). Data analysis displayed that Vd991 inoculation significantly increased GhACS2 or GhACS6 expression in roots during 48 h, especially at 6 h (Fig. 2A), which implied that the activation of GhACS2 or GhACS6 may be involved in early responses of cotton to V. dahliae infection.
To investigate this involvement, GhACS2 and GhACS6 expression in YZ1 seedlings was monitored using qRT-PCR. With no V. dahliae inoculation, GhACS2 and GhACS6 transcript levels were low (Fig. 2B). In response to V. dahliae inoculation, GhACS2 and GhACS6 transcripts were significantly increased in roots, stems and leaves, especially roots and stems (Fig. 2B), with the GhACS2 expression levels in YZ1 plants being 5.5, 8.2 and 4.5 times greater, respectively, than in the control without Vd991 inoculation (Fig. 2B), whereas the GhACS6 expression levels in YZ1 plants were 6.8, 7.8 and 4.0 times greater, respectively, than in the control (Fig. 2B). Thus, Vd991 inoculation induced GhACS2 and GhACS6 expression in cotton plants.
To determine the roles of GhACS2 and GhACS6 in cotton defenses, the GhACS2- and GhACS6-overexpression transgenic lines were created, and five GhACS2-OE and six GhACS6-OE transgenic lines were obtained, respectively. Of them, GhACS2-OE(#1) and GhACS6-OE(#1) had relatively high GhACS2 and GhACS6 expression levels, respectively, and thus were selected for further research (Fig. 2C and 2D). ACC content in GhACS2-OE(#1) and GhACS6-OE(#1) plants with or without Vd991 inoculation was analyzed, and results were as follows: under no Vd991 inoculation conditions, ACC contents in roots of GhACS2-OE(#1) (58.1 ± 6.03 ng/g) and GhACS6-OE(#1) (42.6 ± 3.52 ng/g) were slightly greater than that of YZ1 (32.2 ± 2.12 ng/g). At 16 days after Vd991 inoculation, the ACC contents in roots of GhACS2-OE(#1) (118.5 ± 7.32 ng/g) and GhACS6-OE(#1) (89.2 ± 6.24 ng/g) were significantly greater than that of YZ1 (57.1 ± 4.08 ng/g) (Fig. 2E). Similar scenario occurred in both stems and leaves (Fig. S4). Evidently, ACC accumulation was significantly increased in GhACS2-OE(#1) or GhACS6-OE(#1) plants.
The YZ1 plants were set as the control, Verticillium wilt symptoms caused by Vd991 infection for 16 days were investigated in ACC-accumulated plants. The survey result as follows: (1) the cotyledons of these ACC-accumulated plants exhibited slight wilting and chlorotic symptoms (Fig. 3A, left panel); (2) fungal recovery assays suggested that there were more V. dahliae colonies in stems of YZ1 than in both GhACS2-OE(#1) and GhACS6-OE(#1) (Fig. 3A, right panel); (3) the browning of vascular tissues was more severe in YZ1 plants than in GhACS2-OE(#1) and GhACS6-OE(#1) plants (Fig. 3A, middle panel); (4) the disease indexes were significantly lower in GhACS2-OE(#1) (47.83 ± 3.39%) and GhACS6-OE(#1) (53.28 ± 2.30%) than in YZ1 (68.47 ± 2.19%) (Fig. 3B); (5) V. dahliae biomasses in the stems of GhACS2-OE(#1) and GhACS6-OE(#1) plants were 36% and 29% lower than in YZ1 plants (Fig. 3C). Evidently, the overexpression of GhACS2 and GhACS6 not only increased ACC accumulation, but also improved cotton resistance to Vd991 infection.
GhACS2/6 overexpression improved cotton resistance to V. dahliae infection in the artificial Verticillium wilt nursery
Under the strong infection conditions of open-field Verticillium wilt nursery, we further compared the resistance of GhACS2-OE(#1) and GhACS6-OE(#1) plants to V. dahliae with that of YZ1 in 2019 and 2020 years. Observations indicated: (1) the susceptible YZ1 plants showed typical leaf wilting (Fig. 4A) and serious vascular browning (Fig. 4B) symptoms, but GhACS2-OE(#1) and GhACS6-OE(#1) plants were alleviated (Fig. 4A and 4B); (2) the disease indexes of GhACS2-OE(#1) (2019: 54.86 ± 3.18%; 2020: 58.73 ± 2.18%) and GhACS6-OE(#1) (2019: 53.62 ± 4.19%; 2020: 61.73 ± 3.80%) were lower than those of YZ1 plants (2019: 68.26 ± 2.98%; 2020: 71.43 ± 1.04%) (Fig. 4C); (3) V. dahliae biomasses in stems also decreased in GhACS2-OE(#1) (decreased by 33% in 2019; decreased by 29% in 2020) and GhACS6-OE(#1) (decreased by 29% in 2019; decreased by 25% in 2020) compared with in YZ1 (Fig. 4D). Data suggest that the activation of GhACS2 and GhACS6 and ACC accumulation improved the resistance of cotton to V. dahliae infection.
ACC treatment or ACC accumulation impeded Vd991 colonization and propagation in cotton root tissues
To explain the mechanisms behind ACC’s effects on cotton resistance to V. dahliae, we examined whether ACC affected colonization or diffusion of V. dahliae in root tissues. In no ACC treatment, the GFP-marked V. dahliae stain showed fluorescence emissions in YZ1 roots during inoculation for 12 h. This scenario was similar to those of previous reports [26, 31]. After more time (24, 48 and 72 h), the GFP-marked Vd991 colonization was extended and diffused. For example, the GFP fluorescence intensity at 72 h was 2.31 times greater than that at 12 h (Fig. 5A and 5B). However, in ACC-treated root tissues, there was less of an increase in fluorescence intensity. For example, the GFP fluorescence intensity from ACC-treated roots was 0.67 times that from untreated roots at 72 h (Fig. 5B), which indicated that the ACC treatment reduced V. dahliae conidia in cotton root tissues. In addition, the fungal biomass assay indicated that the ACC treatment reduced the V. dahliae biomass in YZ1 root tissues. For example, V. dahliae inoculation for 72 h, the fungal biomass in ACC-treated root tissues was approximately 0.55 times that of the untreated root tissues (Fig. 5C). That is to say, ACC treatment hindered the V. dahliae invasion of cotton root tissues.
We further investigated how endogenous ACC accumulations affected V. dahliae colonization or reproduction of roots. Confocal microscopic scanning showed that the GFP fluorescence caused by ‘Vd991-GFP’ inoculation was more intense in YZ1 roots than in GhACS2-OE(#1) and GhACS6-OE(#1) root tissues (Fig. 6A). A quantitative analysis showed that, Vd991-GFP inoculation for 72 h, the GFP fluorescence intensity in YZ1 root tissues was approximately 1.45 and 1.31 times greater than those in GhACS2-OE(#1) and GhACS6-OE(#1) root tissues, respectively (Fig. 6B). In addition, the relative fungal biomass in the YZ1 root tissues was approximately 1.96 and 1.64 times greater than those of GhACS2-OE(#1) and GhACS6-OE(#1) root tissues, respectively (Fig. 6C). These data suggested that endogenous ACC accumulations impede V. dahliae colonization of cotton root tissues.
ACC increased the SA-dependent resistance of cotton to V. dahliae infection
It is necessary to determine how ACC affects the SA-dependent resistance of cotton to V. dahliae infection, because SA plays key roles in resisting fungal infections [1, 2]. Because SA production depends on the expression of EDS1 and PAD4 genes after V. dahliae infection [6], we investigated how ACC affected their expression. With no Vd991 infection, ACC treatment increased EDS1 and PAD4 expression (Fig. S5A) and SA content (Fig. S5B). However, ACC significantly increased EDS1 and PAD4 expression (Fig. S5A) and SA production (Fig. S5B) in the root tissues infected by Vd991 stain, compared with that in the untreated and uninfected control.
We further detected how ACC-accumulated plants affected SA production and signaling. With no Vd991 infection, EDS1 and PAD4 expression (Fig. 7A) and SA production (Fig. 7B) were no significant difference between the YZ1 control and the transgenic GhACS2-OE(#1) and GhACS6-OE(#1) plants. In contrast, at 72 h after Vd991 inoculation, EDS1 and PAD4 expression was significantly higher in root tissues of GhACS2-OE(#1) and GhACS6-OE(#1) than that in YZ1 (Fig. 7A). Meantime, the SA contents in GhACS2-OE(#1) (2.46 ± 0.11 µg/g) and GhACS6-OE(#1) (2.28 ± 0.11 µg/g) roots infected by Vd991 were approximately 1.87 and 1.74 times greater, respectively, than that of YZ1 (1.31 ± 0.08 µg/g) root tissues (Fig. 7B).
Because SA induces cotton resistance to fungal infection by activating NPR1, PR1 and PR5 expression [1, 7], we further detected their gene expression levels. Without a Vd991 infection, the expression levels of NPR1, PR1 and PR5 in roots did not significantly change when exposed to ACC treatments (Fig. S5A). However, in ACC-treated YZ1 root tissues, NPR1, PR1 and PR5 expression was significantly increased after Vd991 infection for 72 h (Fig. S5A). Speak specifically, the expression levels of NPR1, PR1 and PR5 were 2.68, 4.37 and 4.82 times higher in root tissues of GhACS2-OE(#1), respectively, and 3.08, 3.28 and 3.76 times higher in root tissues of GhACS6-OE(#1), respectively, than that in YZ1 root tissues (Fig. 7A). These data indicated that the increase in the ACC content of these transgenic cotton plants enhanced the SA-dependent resistance to Vd991 infection.