Citrus CsACD2 Is a Target of Candidatus Liberibacter Asiaticus in Huanglongbing Disease

Abstract

Citrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (Las), is one of the most destructive citrus diseases worldwide, yet how Las causes HLB is poorly understood. Here we show that a Las-secreted protein, SDE15 (CLIBASIA_04025), suppresses plant immunity and promotes Las multiplication. Transgenic expression of SDE15 in Duncan grapefruit (Citrus 3 paradisi) suppresses the hypersensitive response induced by Xanthomonas citri ssp. citri (Xcc) and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the Xanthomonas vesicatoria effector protein AvrBsT in Nicotiana benthamiana, suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis (Arabidopsis thaliana) ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on CsACD2, and overexpression of CsACD2 in citrus suppresses plant immunity and promotes Las multiplication, phenocopying overexpression of SDE15. Identification of CsACD2 as a susceptibility target has implications in genome editing for novel plant resistance against devastating HLB.

Genomic insights into citrus domestication and its important agronomic traits

“… Gene details Identified/cloned Function study Target trait Reference 1 Papain-like cysteine protease Sweet orange – Citrus Huanglongbing Clark et al. (2018) 2 Accelerated cell death 2 Sweet orange Duncan grapefruit Pang et al. (2020) 3 2-oxoglutarate and Fe(II)-dependent oxygenase gene US-897 ( Citrus reticulata × Poncirus trifoliata ) – Albrecht and Bowman (2011) 5 PtCDR2 and PtCDR8 P. trifoliata – Rawat et al.…”

A Sec-Dependent Secretory Protein of the Huanglongbing-Associated Pathogen Suppresses Hypersensitive Cell Death in Nicotiana benthamiana

“…The N-terminal GFP-tagged m4405 was detected under confocal fluorescence microscopy. As shown in Figure 3B, m4405-GFP was predominately colocalized with the plasma membranal marker pm-rk CD3-1007 (Nelson et al, 2007), indicating the plasma membrane localization of m4405 in plants. Using the same strategy, we observed that CLIBASIA_04405 shared a similar subcellular localization in N. benthamiana cells with m4405 ( Figure 3B).…”

“…In contrast, the pathogens, including bacteria, fungi, oomycetes, and nematodes, often secrete an arsenal of effectors that contribute to cell death suppression to enable their successful infection (da Cunha et al, 2007;Dou and Zhou, 2012;Goverse and Smant, 2014;Lo Presti et al, 2015). In CLas, the mature forms of two Sec-dependent secretory proteins CLIBASIA_03875 and CLIBASIA_04025, termed m3875 and SDE15, have been determined as PCD suppressors (Zhang et al, 2019;Pang et al, 2020). Herein, we demonstrated that m4405, the mature form of the Sec-dependent secretory protein CLIBASIA_04405, also inhibited hypersensitive cell death.…”

“…The coding sequences of CLIBASIA_04405 and m4405 amplified with the specific primers ( Supplementary Table 1) were inserted into pCAMBIA1300-35S-GFP digested with Kpn I and Xho I. The resulting p4405-GFP and pm4405-GFP were individually introduced into Agrobacterium tumefaciens EHA105, followed by co-infiltration with agrobacterial cells carrying a plasma membrane marker pm-rk CD3-1007 (Nelson et al, 2007) into leaves of the 4-week-old N. benthamiana, as previously described (Van der Hoorn et al, 2000). At 60 h post inoculation (hpi), the infiltrated patches were visualized for GFP fluorescence under a LSM700 confocal microscope (Zeiss, Germany).…”

“…A significant number of CLas-encoded proteins have been experimentally validated to be Sec-dependent secretory proteins (Prasad et al, 2016;Zhang et al, 2019), and their potential roles in CLas pathogenesis are beginning to be revealed. For example, CLIBASIA_03875 and CLIBASIA_04025 suppress programmed cell death (PCD) (Zhang et al, 2019;Pang et al, 2020), CLIBASIA_05315 targets the chloroplasts and induces cell death in Nicotiana benthamiana (Pitino et al, 2016(Pitino et al, , 2018 as well as physically interacting with the papain-like cysteine proteases (PL) and inhibiting their protease activity in citrus (Clark et al, 2018). In addition, a subset of Sec-dependent secretory proteins were deduced to be candidate effectors that play roles during CLas infection using a Tobacco mosaic virusbased expression system or a strategy for the analysis of temporal and spatial gene expression (Shi et al, 2019;Ying et al, 2019;Li et al, 2020).…”