Preharvest UV-C Hormesis Induces Key Genes Associated With Homeostasis, Growth and Defense in Lettuce Inoculated With Xanthomonas campestris pv. vitians

Abstract

Preharvest application of hormetic doses of ultraviolet-C (UV-C) generates beneficial effects in plants. In this study, within 1 week, four UV-C treatments of 0.4 kJ/m2 were applied to 3-week-old lettuce seedlings. The leaves were inoculated with a virulent strain of Xanthomonas campestris pv. vitians (Xcv) 48 h after the last UV-C application. The extent of the disease was tracked over time and a transcriptomic analysis was performed on lettuce leaf samples. Samples of lettuce leaves, from both control and treated groups, were taken at two different times corresponding to T2, 48 h after the last UV-C treatment and T3, 24 h after inoculation (i.e., 72 h after the last UV-C treatment). A significant decrease in disease severity between the UV-C treated lettuce and the control was observed on days 4, 8, and 14 after pathogen inoculation. Data from the transcriptomic study revealed, that in response to the effect of UV-C alone and/or UV-C + Xcv, a total of 3828 genes were differentially regulated with fold change (|log2-FC|) > 1.5 and false discovery rate (FDR) < 0.05. Among these, of the 2270 genes of known function 1556 were upregulated and 714 were downregulated. A total of 10 candidate genes were verified by qPCR and were generally consistent with the transcriptomic results. The differentially expressed genes observed in lettuce under the conditions of the present study were associated with 14 different biological processes in the plant. These genes are involved in a series of metabolic pathways associated with the ability of lettuce treated with hormetic doses of UV-C to resume normal growth and to defend themselves against potential stressors. The results indicate that the hormetic dose of UV-C applied preharvest on lettuce in this study, can be considered as an eustress that does not interfere with the ability of the treated plants to carry on a set of key physiological processes namely: homeostasis, growth and defense.

Keywords: bacterial disease; cell homeostasis; defense mechanisms; eustress; leaf spot disease; lettuce; physiological processes; plant growth.

FIGURE 1

Effect of UV-C treatments on AUDPC (area under the disease progress curve). Treatments (Trt): Cont (control), UV (1.6 kJ/m²). Two-way ANOVA indicates a significant effect of treatments, assessment days (0, 4, 8, and 14) for BLS (bacterial leaf spot) and their interaction. AUDPC means are statistically different at P < 0.05 according to the Duncan’s multiple range test, n = 9, ^***P < 0.001. Different letters indicate a significant difference between treatments at the same day or on different days (4, 8, and 14). The vertical bars show the standard deviation.

FIGURE 2

Genes related to metal transport differentially expressed in response to UV and Xcv inoculation. Test of hierarchical DEG (HCA) groupings. The colors reflect the level of relative gene expression. The columns and rows represent the samples and ID_genes associated with the name/function of the corresponding proteins that were grouped according to their relative expression profile, respectively. C-48 h, control corresponding to treated 48 h after UV; UV-48 h, UV after 48 h treatment; C-72 h, control corresponding to treated 72 h after UV; UV-72 h, UV after 72 h treatment; Ci-24 h, control 24 h after inoculation; UVi-24h, UV 24 h after inoculation. The genes were filtered out with a threshold |log2-FC| > 1.5 and FDR < 0.05.

FIGURE 3

Primary metabolism-related genes differentially expressed in response to the influence of UV and Xcv inoculation. Test of hierarchical DEG (HCA) groupings. The colors reflect the level of relative gene expression. The columns and rows represent the samples and ID_genes associated with the name/function of the corresponding protein that were grouped according to their relative expression profile, respectively. C-48 h, control corresponding to treated 48 h after UV; UV-48 h, UV after 48 h treatment; C-72 h, control corresponding to treated 72 h after UV; UV-72 h, UV after 72 h treatment; Ci-24 h, control 24 h after inoculation; UVi-24 h, UV 24 h after inoculation. The genes were filtered out with a threshold |log2-FC| > 1.5 and FDR < 0.05.

FIGURE 4

Genes related to defense mechanisms differentially expressed in response to UV and Xcv inoculation. Test of hierarchical DEG (HCA) groupings. The colors reflect the level of relative gene expression. The columns and rows represent the samples and ID_genes associated with the name/function of the corresponding protein that were grouped according to their relative expression profile, respectively. C-48 h, control corresponding to treated 48 h after UV; UV-48 h, UV after 48 h treatment; C-72 h, control corresponding to treated 72 h after UV; UV-72 h, UV after 72 h treatment; Ci-24 h, control 24 h after inoculation; UVi-24 h, UV 24 h after inoculation. The genes were filtered out with a threshold |log2-FC| > 1.5 and FDR < 0.05.

FIGURE 5

Comparison of gene expression levels between RNA-seq and qPCR. On the X-axis, we have qPCR data coupled to RNA-seq data for each gene and for each treatment: UV-48 h, UV after 48 h treatment; UV-72 h, UV after 72 h treatment; UVi-24 h, UV 24 h after inoculation. C-48h (Control corresponding to treated 48 h after UV) was used as a control.

FIGURE 6

Representative model of the regulation mechanisms induced in lettuce by the preharvest application of hormetic doses of UV-C and inoculation with the pathogen Xcv. Thick-bordered rectangular boxes: Physiological (or biological) processes; dashed rectangular boxes: Function or molecules associated with genes as reported in the literature. Thin-bordered ellipses: genes affected by UV-C alone; thick-bordered ellipses: genes affected by UV-C + Xcv infection. The pointed arrows indicate activation reactions and the blunt arrows indicate inhibition reactions. The numbers in brackets in the rectangular boxes represent the reference source(s). (1) Cheng et al. (2018); (2) Kim et al. (2007); (3) Wang et al. (2016); (4) Pushpa et al. (2014); (5) Savidge et al. (2002); (6) Lokdarshi et al. (2016); (7) Gollhofer et al. (2014); (8) Cassin et al. (2009); (9) Le et al. (2016); (10) Kato and Sakamoto (2018); (11) Yan et al. (2016); (12) Lunn et al. (2014); (13) Shen et al. (2017); (14) Doan et al. (2009); (15) Nayeri and Yarizade (2014); (16) Dey and Vlot (2015); (17) Zhang and Liu (2015); (18) Ma et al. (2005); (19) Ziegler and Facchini (2008); (20) Sung et al. (2011); (21) Höfer et al. (2013); (22) Noda et al. (2013); (23) Zhou et al. (2014); (24) Bundó and Coca (2017); (25) Li et al. (2020); (26) van Engelen et al. (1993); (27) Kimura and Tanaka (2010); (28) Ashrafi-Dehkordi et al. (2018); (29) Hauser et al. (2015); (30) Li et al. (2018); (31) Chaiwanon et al. (2016); (32) Qiao et al. (2019); (33) Tan and Kamada (2000); (34) Skaar et al. (2013); (35) Ban et al. (2020); (36) Han et al. (2013); (37) Zhao et al. (2018); (38) Lannoo and Van Damme (2014); (39) De Storme and Geelen (2014); (40) Zhu et al. (2016); (41) Lionetti et al. (2017); (42) Niu and Wang (2020); (43) Jamieson et al. (2018); (44) Dutton et al. (2019).