Overexpressing CsGH3.1 and CsGH3.1L reduces susceptibility to Xanthomonas citri subsp. citri by repressing auxin signaling in citrus (Citrus sinensis Osbeck)

The auxin early response gene Gretchen Hagen3 (GH3) plays dual roles in plant development and responses to biotic or abiotic stress. It functions in regulating hormone homeostasis through the conjugation of free auxin to amino acids. In citrus, GH3.1 and GH3.1L play important roles in responding to Xanthomonas citri subsp. citri (Xcc). Here, in Wanjingcheng orange (C. sinensis Osbeck), the overexpression of CsGH3.1 and CsGH3.1L caused increased branching and drooping dwarfism, as well as smaller, thinner and upward curling leaves compared with wild-type. Hormone determinations showed that overexpressing CsGH3.1 and CsGH3.1L decreased the free auxin contents and accelerated the Xcc-induced decline of free auxin levels in transgenic plants. A resistance analysis showed that transgenic plants had reduced susceptibility to citrus canker, and a transcriptomic analysis revealed that hormone signal transduction-related pathways were significantly affected by the overexpression of CsGH3.1 and CsGH3.1L. A MapMan analysis further showed that overexpressing either of these two genes significantly downregulated the expression levels of the annotated auxin/indole-3-acetic acid family genes and significantly upregulated biotic stress-related functions and pathways. Salicylic acid, jasmonic acid, abscisic acid, ethylene and zeatin levels in transgenic plants displayed obvious changes compared with wild-type. In particular, the salicylic acid and ethylene levels involved in plant resistance responses markedly increased in transgenic plants. Thus, the overexpression of CsGH3.1 and CsGH3.1L reduces plant susceptibility to citrus canker by repressing auxin signaling and enhancing defense responses. Our study demonstrates auxin homeostasis’ potential in engineering disease resistance in citrus.

cultivars, including orange, grapefruit, lime, lemon, pomelo and citrus rootstock [1] . 40 The canker's development includes the initial appearance of oily looking spots, 41 usually on the abaxial leaf surface, outbursts of white or yellow spongy pustules and 42 finally the formation of brown corky cankers [2] . Pustule formation (excessive cell 43 division) in the infected tissues plays a vital role in citrus canker development and 44 pathogen spread [1,[3][4][5] . The inhibition or disruption of pustule development can 45 efficiently repressed pathogen spread and even confer plant resistance to citrus canker 46 [6,7] , indicating that the manipulation of pustule development is a potential strategy for  Auxin, a critical plant hormone that controls a range of plant growth and 52 developmental processes, including cell division and expansion, has long been 53 recognized as a regulator of plant defenses [8,9] . The effector AvrRpt2 from and JA was simultaneously measured as described by [29,30] . In brief, Tissue samples 141 (1 g fresh weight) were frozen in liquid nitrogen, ground to a fine powder, extracted 142 with 80% methanol overnight and then centrifuged at 13,000 ×g for 10 min. The were placed in a gas-tight container equipped with septa, and 1 mL of headspace gas 149 was sampled using a gas syringe [31] . The ethylene production was measured using gas 150 chromatograph. The test was repeated three times.

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The disease resistance assay for transgenic plants against citrus canker was performed 153 according to Peng et al. [6] . A Xcc strain, XccYN1 [6] , was used to investigate plant 154 disease resistance. Three mature healthy leaves per plant were tested. In total, 24 155 punctures were made per leaf with a needle containing the bacterial suspension (0.5 × 156 10 5 CFU ml −1 ). The inoculated leaves were maintained at 28°C under a 16-h light/8-h 157 dark photoperiod with 45 μmol m −2 s −1 illumination and 60% relative humidity.

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Photographs were taken at 10 dpi. The area of all diseased spots was assessed with surrounding the 24 punctures on three leaves using the rating index described by Peng 162 et al. [6] . The test was repeated three times. were estimated using the FPKM method [33] . A differential expression analysis 182 between transgenic lines and WT was performed using the DESeq R package 1.10.1 183 [34] . Genes with adjusted P-values < 0.05 found by DESeq were assigned as 184 differentially expressed genes (DEGs). A GO enrichment analysis of the DEGs was 185 performed using the GOseq R package [35] . A KEGG pathway enrichment analysis of 186 DEGs was performed using KOBAS software [36] .

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After one year, lines 1-3 and 1-4 died. After two years, transgenic plants displayed a 214 bushy dwarf phenotype with smaller, drooping and upward curling leaves and branch 215 softening and drooping ( Fig. 1b and Fig. S4). Moreover, transgenic leaves were 216 significantly thinner, and both their longitudinal and transverse diameters were 217 significantly shorter compared with WT ( Fig. 1c-

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To investigate the auxin content, the free IAA level in each transgenic line was 230 measured. The free IAA levels in the 1-5, 1-8, 1-9, L-2, L-5 and L-6 transgenic lines 231 were significantly lower than in WT plants before exposure to Xcc (Fig. 2). Other

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To further survey the pathways or functions that were affected by the DEGs in  Auxin-related genes 312 We investigated the DEGs related to auxin homeostasis, perception and signaling in  (Table 1). All 12 Aux/IAA family members, a group of auxin-induced 317 genes, showed significantly downregulated expression levels in the transgenic plants. 318 In addition, two of four SAUR-like auxin-responsive protein family members (ARG7), 319 and one auxin response factor (ARF10) also showed significantly downregulated 320 expression levels in the transgenic plants.

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Data presented in Table 3 show that 11 cell wall-related genes were differentially   and L-5 transgenic lines (Fig. 7). The SA and ET contents in both 1-9 and L-5 Auxin is believed to act as a pathogenic factor in pustule formation during citrus 451 canker development [5,16,26]