Wang, Xuefeng

Abstract Autophagy functions in plant host immunity responses to pathogen infection. The molecular mechanisms and functions used by the citrus Huanglongbing (HLB)-associated intracellular bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) to manipulate autophagy are unknown. We identified a CLas effector, SDE4405 (CLIBASIA_04405), which contributes to HLB progression. ‘Wanjincheng’ orange (Citrus sinensis) transgenic plants expressing SDE4405 promotes CLas proliferation and symptom expression via suppressing host immunity responses. SDE4405 interacts with the ATG8-family of proteins (ATG8s), and their interactions activate autophagy in Nicotiana benthamiana. The occurrence of autophagy is also significantly enhanced in SDE4405-transgenic citrus plants. Interrupting NbATG8s-SDE4405 interaction by silencing of NbATG8c reduces Pseudomonas syringae pv. tomato strain DC3000ΔhopQ1-1 (Pst DC3000ΔhopQ1-1) proliferation in N. benthamiana, and transient overexpression of CsATG8c and SDE4405 in citrus promotes Xanthomonas citri subsp. citri (Xcc) multiplication, suggesting that SDE4405-ATG8s interaction negatively regulates plant defense. These results demonstrate the role of the CLas effector protein in manipulating autophagy, and provide new molecular insights into the interaction between CLas and citrus hosts.

AbstractCitrus Huanglongbing (HLB), caused by Candidatus Liberibacter asiaticus (CaLas), is the most serious disease worldwide. CaLasSDE460 was previously characterized as a potential virulence factor of CaLas. However, the function and mechanism of CaLasSDE460 involved in CaLas against citrus is still elusive. Here, we showed that transgenic expression of CaLasSDE460 in Wanjincheng oranges (C. sinensis Osbeck) contributed to the early growth of CaLas and the development of symptoms. When the temperature increased from 25 °C to 32 °C, CaLas growth and symptom development in transgenic plants were slower than those in WT controls. RNA-seq analysis of transgenic plants showed that CaLasSDE460 affected multiple biological processes. At 25 °C, transcription activities of the “Protein processing in endoplasmic reticulum” and “Cyanoamino acid metabolism” pathways increased while transcription activities of many pathways decreased at 32 °C. 124 and 53 genes, separately annotated to plant-pathogen interaction and MAPK signaling pathways, showed decreased expression at 32 °C, compared with these (38 for plant-pathogen interaction and 17 for MAPK signaling) at 25 °C. Several important genes (MAPKKK14, HSP70b, NCED3 and WRKY33), remarkably affected by CaLasSDE460, were identified. Totally, our data suggested that CaLasSDE460 participated in the pathogenesis of CaLas through interfering transcription activities of citrus defense response and this interfering was temperature-dependent. Graphical Abstract

Citrus Huanglongbing (HLB) is the most destructive citrus disease worldwide, mainly caused by ‘Candidatus Liberibacter asiaticus’ (CLas). It encodes a large number of Sec-dependent effectors that contribute to HLB progression. In this study, an elicitor triggering ROS burst and cell death in Nicotiana benthamiana, CLIBASIA_04425 (CLas4425), was identified. Of particular interest, its cell death-inducing activity is associated with its subcellular localization and the cytoplasmic receptor Botrytis-induced kinase 1 (BIK1). Compared with CLas infected psyllids, CLas4425 showed higher expression level in planta. The transient expression of CLas4425 in N. benthamiana and its overexpression in Citrus sinensis enhanced plant susceptibility to Pseudomonas syringae pv. tomato DC3000 ΔhopQ1-1 and CLas, respectively. Furthermore, the salicylic acid (SA) level along with the expression of genes NPR1/EDS1/NDR1/PRs in SA signal transduction was repressed in CLas4425 transgenic citrus plants. Taken together, CLas4425 is a virulence factor that promotes CLas proliferation, likely by interfering with SA-mediated plant immunity. The results obtained facilitate our understanding of CLas pathogenesis.

Huanglongbing (HLB) is a global devastating citrus disease that is mainly caused by “Candidatus Liberibacter asiaticus” (CLas). It is mostly transmitted by the insect Asian citrus psyllid (ACP, Diaphorina citri) in a persistent and proliferative manner. CLas traverses multiple barriers to complete an infection cycle and is likely involved in multiple interactions with D. citri. However, the protein–protein interactions between CLas and D. citri are largely unknown. Here, we report on a vitellogenin-like protein (Vg_VWD) in D. citri that interacts with a CLas flagellum (flaA) protein. We found that Vg_VWD was upregulated in CLas-infected D. citri. Silencing of Vg_VWD in D. citri via RNAi silencing significantly increased the CLas titer, suggesting that Vg_VWD plays an important role in the CLas–D. citri interaction. Agrobacterium-mediated transient expression assays indicated that Vg_VWD inhibits BAX- and INF1-triggered necrosis and suppresses the callose deposition induced by flaA in Nicotiana benthamiana. These findings provide new insights into the molecular interaction between CLas and D. citri.

Abstract Citrus huanglongbing (HLB), associated with the unculturable phloem‐limited bacterium “ Candidatus Liberibacter asiaticus” ( C Las), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of C Las remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the C Las genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis . The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H 2 O 2 and O 2 − ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri . This study reveals a novel mechanism underlying how C Las uses a prophage‐encoded effector, AGH17488, to target a reactive oxygen species accumulation‐related gene, APX6 , in the host to facilitate its infection.

Citrus Huanglongbing (HLB), known as the most economically devastating disease in citrus industry, is mainly caused by phloem-restricted Gram-negative bacterium “Candidatus Liberibacter asiaticus” (CLas). To date, CLas is still unculturable in vitro, which has been dramatically delaying the research on its pathogenesis, and only few Sec-dependent effectors (SDEs) have been identified to elucidate the pathogenesis of CLas. Here, we confirmed that a CLas-secreted Sec-dependent polypeptide, namely SECP8 (CLIBASIA_05330), localized in nucleus, cytoplasm and cytoplasmic membrane, and showed remarkably higher transcript abundance in citrus than in psyllids. Potato virus X (PVX)-mediated transient expression assays indicated that mSECP8 (the mature form of SECP8) suppressed pro-apoptotic mouse protein BAX and Phytophthora infestans elicitin INF1-triggered hypersensitive response (HR) associated phenotypes, including cell death, H2O2 accumulation and callose deposition. Intriguingly, mSECP8 also inhibited SDE1 (CLIBASIA_05315)-induced water-soaked and dwarfing symptoms in Nicotiana benthamiana. In addition, mSECP8 can promote the susceptibility of transgenic Wanjincheng orange (Citrus sinensis) to CLas invasion and further HLB symptom development, and it contributes to the proliferation of Xanthomonas citri subsp. citri (Xcc). Moreover, the expression of ten immunity-related genes were significantly down-regulated in mSECP8 transgenic citrus than those in wide-type (WT) plants. Overall, we propose that mSECP8 may serve as a novel broad-spectrum suppressor of plant immunity, and provide the first evidence counteractive effect among CLas effectors. This study will enrich and provide new evidences for elucidating the pathogenic mechanisms of CLas in citrus host.

Abstract Huanglongbing (HLB), associated with “ Candidatus Liberibacter asiaticus” (CLas), is a globally devastating plant disease. The highly reduced genome of CLas encodes a number of secretory proteins. The conserved prophage‐encoded protein AGH17470 is herein identified as a nonclassical secretory protein. We confirmed that the N‐terminal and C‐terminal sequences jointly determine the secretion of AGH17470. The transient expression of AGH17470 protein in Nicotiana benthamiana caused hypersensitive response (HR) cell death in infiltrated leaves and systemically infected leaves as well as the dwarfing of the entire plant, suggesting that AGH17470 is involved in the plant immune response, growth, and development. Overexpression of AGH17470 in N. benthamiana and citrus plants up‐regulated the transcription of pathogenesis‐related and salicylic acid (SA)‐signalling pathway genes and promoted SA accumulation. Furthermore, transient expression of AGH17470 enhanced the resistance of sweet orange to Xanthomonas citri subsp. citri . To our knowledge, AGH17470 is the first prophage‐encoded secretory protein demonstrated to elicit an HR and induce a strong plant immune response. The findings have increased our understanding of prophage‐encoded secretory protein genes, and the results provide clues as to the plant defence response against CLas.

Citrus huanglongbing (HLB) is present in 10 provinces in China and is associated with ‘Candidatus Liberibacter asiaticus’ (CLas), which is transmitted by the Asian citrus psyllid (Diaphorina citri, ACP). To date, HLB and ACP have expanded to Yibin city of Sichuan Province, posing an imminent threat to the citrus belt of the upper and middle reaches of the Yangtze River, an important late-maturing citrus-producing area in China. To understand the epidemiological route of CLas and ACP in newly invaded regions of Sichuan and thereby better establish an HLB interception zone ranging from Leibo to Yibin, we evaluated the molecular variability of 19 CLas draft genomes from citrus or dodder (Cuscuta campestris). They include three type-specific prophage loci, three variable number tandem repeat loci, a miniature inverted-repeat transposable element, and population diversity of 44 ACP mitochondrial genomes. The results indicated that CLas isolates in the newly invaded area (Pingshan) were more diverse than those in the HLB endemic areas (Leibo and Ningnan). Phylogenetic analysis based on mitochondrial genomes demonstrated that ACPs in Leibo, Pingshan, and Xuzhou (rural areas) represent a new mitochondrial group (MG4), distinguished by the three unique single-nucleotide polymorphisms in cox1, nad4, and cytb. However, the ACPs sampled from the urban areas of Cuiping and Xuzhou belonged to the southeastern China group (MG2-1). Altogether, our study revealed multiple sources of ACP and CLas in the HLB interception zone and proposed their transmission route. This study contributes to the formulation of precise HLB prevention and control strategies in the HLB interception zone in Sichuan and could be useful for HLB management efforts in other regions.