as CsACD2 is a target of Candidatus Liberibacter asiaticus in Huanglongbing disease

One-sentence Candidatus Liberibacter asiaticus targets citrus CsACD2 to promote devastating Huanglongbing ABSTRACT 30 Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the 31 most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here 32 we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity 33 and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit ( Citrus 34 × paradisi ) suppresses the hypersensitive response (HR) induced by Xanthomonas citri subsp. 35 citri ( Xcc ) and reduces the expression of immunity-related genes. SDE15 also suppresses the HR 36 triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana , 37 suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with 38 the citrus protein CsACD2, a homologue of Arabidopsis ACCELERATED CELL DEATH 2 39 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2 , and overexpression 40 of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying 41 overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in 42 genome editing for novel plant resistance against devastating HLB. 43 44 45 , GST1 , WRKY22 , WRKY29 , PR1 , PR3 and PR5 in the CsACD2 -RNAi transgenic citrus was compared to the wild type Duncan grapefruit. The housekeeping gene encoding glyceraldehyde-3- phosphate dehydrogenase-C (GAPDH-C) was used as an endogenous control. Error standard error of mean (n=4). significant differences in different types of samples by one-way ANOVA followed by least significant difference post hoc test (p-Value < 0.05). All experiments were repeated three times with the similar results. Targeted promoter editing for rice resistance to Xanthomonas oryzae pv. oryzae reveals differential activities for SWEET14-inducing TAL effectors.


SDE15 is detected in the phloem sap
Sequence analysis of SDE15 did not reveal insights regarding its function except for the presence 117 of a typical signal peptide (amino acids 1-22) predicted by SignalP v4.1. To determine whether 118 SDE15 is secreted into the cytosol of phloem cells, and may be detected in the phloem sap of 119 Las-infected plants, we used the SDE15-specific antibody to examine the presence of SDE15 in 120 infected citrus plants. As shown in Fig. 3A, SDE15 is clearly detected in the phloem sap extract 121 of Las-infected citrus bark but not in that of healthy citrus by western blot analysis. PCR analysis 122 using Las-specific primers showed the presence of Las in the bark samples, but not in the phloem 123 sap extract samples (Fig. 3A), suggesting that SDE15 detected in the phloem sap is not directly 124 associated with Las cells per se, but is secreted into phloem cells. 125 We next analyzed the expression pattern of SDE15 in Las-infected citrus and psyllids using 126 reverse transcription quantitative PCR (RT-qPCR). SDE15 showed a higher expression level in 127 citrus than in psyllids (Fig. 3B). Furthermore, SDE15 expression level was higher in the early 128 stage (asymptomatic) than the later stage (symptomatic) of Las infection (Fig. 3C), indicating 129 that SDE15 likely functions at a relatively early stage of Las infection in citrus before the 130 appearance of visible HLB symptoms. 6 immunity via protein-protein interactions. To identify the targets of SDE15, we performed yeast 135 two-hybrid (Y2H) screening with SDE15 without its signal peptide using cDNA libraries 136 generated with mRNAs isolated from the Valencia sweet orange (Citrus × sinensis) at different 137 Las infection stages (healthy (H), asymptomatic (AS), and symptomatic (S)). We identified 20 138 SDE15-interacting proteins in the H library, 60 in the AS library, and 6 in the S library 139 (Supplementary Tables 1, 2&3). Among the SDE15-interacting proteins identified by Y2H, 140 proteins encoded by Cs1g22670 and Cs8g20660 are potentially involved in plant immunity.

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Cs1g22670 encodes a red chlorophyll catabolite reductase (ACD2, hereinafter referred to as 142 CsACD2) that represses programmed cell death in plants (Yao and Greenberg, 2006); whereas 143 Cs8g20660 encodes a ubiquitin-activating enzyme E1 playing roles in innate immunity 144 (Goritschnig et al., 2007). However, further experiments showed that the putative interaction 145 between Cs8g20660 and SDE15 could not be validated by using pair-wise Y2H assay. On the 146 other hand, the interaction between CsACD2 and SDE15 was further confirmed using pair-wise 147 Y2H assay (Fig. 4A), a glutathione-S-transferase (GST) pull-down assay in vitro (Fig. 4B,) and 148 co-immunoprecipitation assays (CoIP) assay in vivo (Fig. 4C). 149 To define the regions of SDE15 and CsACD2 involved in the interaction, truncated proteins CsACD2 with 71-aa deletion at the N-terminus of CsACD2) were expressed in E. coli, purified 154 and used in GST pull-down assays (Fig. 4 D&E). We found that the RCCR domain of CsACD2 155 was sufficient to interact with GST-SDE15 (Fig. 4D). In addition, deletion of the C-terminus, but 156 not the N-terminus of SDE15 abolished its interaction with CsACD2 (Fig. 4E). 7 Intriguingly, however, we found that transient overexpression of SDE15-YFP protein alone was 165 also sufficient to suppress AvrBsT-elicited HR in N. benthamiana (Fig. 5A), suggesting two 166 possibilities: 1) SDE15 interacts with the endogenous homolog of ACD2 in N. benthamiana 167 (NbACD2 hereinafter); or 2) SDE15 directly interferes with plant immunity without involving 168 ACD2. In support of the former scenario, GST pull-down assays showed that SDE15 interacted 169 strongly with NbACD2 (Fig. 5C). As in the case of CsACD2, the NbACD2-YFP fusion protein 170 alone, when transiently overexpressed, was sufficient to suppress HR induction by AvrBsT (Figs. 171 5D&E, S2). Furthermore, silencing of NbACD2 in N. benthamiana plants abolished the SDE15 172 suppression of AvrBsT-triggered HR (Fig. 6), suggesting that SDE15 suppression of plant 173 immunity is ACD2-dependent. In addition, overexpression of CsACD2 in Duncan grapefruit had 174 a similar effect as overexpression of SDE15 in suppressing the HR induced by XccA w (Fig. 7A,

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B, and C; Fig. S3). Overexpression of CsACD2 suppressed the expression of defense-related 176 genes ( Fig. S4), promoted Las growth (Fig. 7D), and facilitated the multiplication of XccA 306 177 in the CsACD2ox plants than the EV-transgenic Duncan grapefruit plants (Fig. 7E). Thus,178 CsACD2 acts as a bona fide susceptibility target gene for Las.

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Auto-immune response was observed on CsACD2-RNAi transgenic citrus 180 Next, we went on to produce CsACD2-silenced transgenic citrus plants to determine whether 181 such plants would be resistant to Las. A fragment targeting the 2 nd exon and 3ʹ-UTR regions was 182 designed to construct a sense-loop-antisense RNAi vector and transferred into Duncan grapefruit 183 via stable Agrobacterium-mediated transformation. Of five independent shootings with erGFP-184 specific fluorescence obtained in total, chlorosis was observed on four of them (Lines 1, 2, 3, and 185 5), and spontaneous cell death was firstly observed on Lines 3 and 5, then lines 1 and 2 (Fig. 8A). 186 Each of these four transgenic shootings eventually died with the lesions spread over the whole 187 plant.

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RNA samples from the transgenic shootings were collected before they died to detect the 189 expression of CsACD2 and defense-related genes (Fig. 8B) downstream target of SDE15. SDE15 suppression of plant immunity is dependent on CsACD2.

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Overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication.

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Vector construction 256 To generate the SDE15-expressing construct for plant transformation, Las genomic DNA was 257 isolated from HLB diseased citrus leaves by the CTAB method (Allen et al., 2006 and then micro-grafted in vitro onto 1-month-old Carrizo citrange nucellar rootstock seedlings.

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After a month of growth in vitro, the grafted shoots were potted into a peat-based commercial 318 potting medium and acclimated under greenhouse conditions. The EV-transformed citrus were 319 also generated as control for further pathogenicity assay.  photographed at 3, 5, 7, and 9 days post inoculation. Growth curve assays of Xcc 306 were 346 conducted at 0, 1, 3, 5, 7, and 9 days post inoculation.

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The following supplemental materials are available.

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The authors have no conflicts of interest to declare.       Yeast-two hybrid (Y2H) assays using SDE15 without the signal peptide as the bait and full-length CsACD2 protein as the prey. SDE15 fused to the GAL4 DNA binding domain (BD) was expressed in combination with CsACD2 fused to the GAL4 activation domain (AD) in the yeast strain Y2HGold. Strains were grown on double dropout medium (DDO) with -Trp and -Leu and screened on quadruple dropout medium (QDO) with -Trp, -Leu, -Ade and -His supplemented with X-α-Gal and Aureobasidin A (QDO/X/A). The empty BD and AD vectors were used as the negative controls. B. Glutathione-S-transferase (GST) pull-down assay. GST-SDE15 and GST empty vectors were expressed in E. coli, immobilized on glutathione sepharose beads, and incubated with E. coli lysate containing MBP-CsACD2. Total cell extract (Input) and eluted protein (Elute) were immunoblotted using the anti-MBP and anti-GST antibody. C. Co-immunoprecipitation (Co-IP) assay. HA-tagged CsACD2 and eYFP-tagged SDE15 were co-expressed in the leaf of N. benthamiana through agroinfiltration. HA tag and eYFP proteins were also expressed in the leaf of N.benthamiana as controls. Co-IP assays were performed using anti-HA and anti-GFP antibodies to determine associations. D. The RCCR domain of CsACD2 interacts with the SDE15 in E. coli lysate. E. The C-terminal of SDE15 interacts with CsACD2. GST-SDE15, GST-SDE15∆N, GST-SDE15∆C, and GST empty vectors were expressed in E. coli, immobilized on glutathione sepharose beads, and incubated with E. coli lysate containing MBP-CsACD2, MBP-RCCR and free MBP proteins. Total cell extract (Input) and eluted protein (Elute) were immunoblotted using the anti-MBP and anti-GST antibody.
www.plantphysiol.org on November 3, 2020 -Published by Downloaded from Copyright © 2020 American Society of Plant Biologists. All rights reserved.   qRT-PCR analysis of defense related genes. Expression of FRK1, GST1, WRKY22, WRKY29, PR1, PR3 and PR5 in the CsACD2-RNAi transgenic citrus was compared to the wild type Duncan grapefruit. The housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase-C (GAPDH-C) was used as an endogenous control. Error bars indicate standard error of mean (n=4). Letters represent significant differences in different types of samples by one-way ANOVA followed by least significant difference post hoc test (p-Value < 0.05). All experiments were repeated three times with the similar results.