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.
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.
Citrus Huanglongbing (HLB) is the most devastating disease of citrus caused by the Gram-negative phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas). It can be transmitted by the Asian citrus psyllid “Diaphorina citri,” by grafting, and by the holoparasitic dodder. In this study, the non-natural host periwinkle (Catharanthus roseus) was infected via dodder (Cuscuta campestris) from CLas-infected citrus plants, and the asymptomatic leaves (AS) were subjected to transcriptomic and small-RNA profiling. The results were analyzed together with a transcriptome dataset from the NCBI repository that included leaves for which symptoms had just occurred (S) and yellowing leaves (Y). There were 3,675 differentially expressed genes (DEGs) identified in AS, and 6,390 more DEGs in S and further 2109 DEGs in Y. These DEGs were commonly enriched in photosystem, chloroplast, membrane, oxidation-reduction process, metal/zinc ion binding on GO. A total of 14,974 DEGs and 336 DE miRNAs (30 conserved and 301 novel) were identified. Through weighted gene co-expression network and nested network analyses, two critical nested miRNA–mRNA regulatory networks were identified with four conserved miRNAs. The primary miR164-NAC1 network is potentially involved in plant defense responses against CLas from the early infection stage to symptom development. The secondary network revealed the regulation of secondary metabolism and nutrient homeostasis through miR828-MYB94/miR1134-HSF4 and miR827-ATG8 regulatory networks, respectively. The findings discovered new potential mechanisms in periwinkle–CLas interactions, and its confirmation can be done in citrus–CLas system later on. The advantages of periwinkle plants in facilitating the quick establishment and greater multiplication of CLas, and shortening latency for disease symptom development make it a great surrogate for further studies, which could expedite our understanding of CLas pathogenesis.
‘Candidatus Liberibacter asiaticus’ (CLas) is a pathogen causing Huanglongbing (HLB, yellow shoot disease), which is highly destructive to citrus production. The CLas strains harbor prophages. We identified two unique prophages, designated as P-PA19-1 and P-PA19-2, in CLas strain PA19 from Pakistan using next-generation sequencing analysis. P-PA19-1 prophage has high sequence similarity (identity: 78.23%) at the early-gene region of prophage SC1 (Type 1), but it is significantly divergent in the late-gene region (identity: 62.03%). P-PA19-2 was highly similar to SC2 (Type 2) in the late gene region (identity: 97.96%), and also in the early gene region except for a deletion of a 7,179-bp nucleotide sequence that contains a CRISPR/cas system in SC2. Both P-PA19-1 and P-PA19-2 had circular plasmid forms, and only P-PA19-2 was found integrated in the PA19 chromosome. The two new prophages were only found in Pakistani samples. Identification of prophages enhances our understanding of CLas genomic diversity and also the biology and evolution of CLas prophages.
‘Candidatus Liberibacter asiaticus’ (CLas) is an unculturable, phloem-restricted αProteobacteria, associated with citrus Huanglongbing (HLB), which is one of the most destructive diseases in citrus production worldwide. Here, we present the genome sequences of CLas strains PA19 and PA20 from HLB-affected kinnow trees in Multan, Punjab Province, Pakistan. The CLas genomes of PA19 and PA20 comprise 1,224,156 bp and 1,226,225 bp, respectively, with an average GC content of 36.4%. Both harbored the Type 2 prophage. In this study, we report two CLas genomes from Pakistan, which extends the sequence database of CLas and will contribute to CLas biology and HLB management.
Huanglongbing (HLB) is a highly destructive disease of citrus production worldwide. 'Candidatus Liberibacter asiaticus', an unculturable alpha proteobacterium, is a putative pathogen of HLB. Information about the biology and strain diversity of 'Ca. L. asiaticus' is currently limited, inhibiting the scope of HLB research and control.
A genomic region (CLIBASIA_05640 to CLIBASIA_05650) of 'Ca. L. asiaticus' showing hyper-sequence variation or locus mosaicism was identified and investigated using 262 bacterial strains (188 from China and 74 from Florida). Based on the characteristic electrophoretic profiles of PCR amplicons generated by a specific primer set, eight electrophoretic types (E-types) were identified, six E-types (A, B, C, D, E, and F) in China and four E-types (A, C, G, and H) in Florida. The 'Ca. L. asiaticus' strains from China consisted predominately of E-type A (71.3%) and E-type B (19.7%). In contrast, the 'Ca. L. asiaticus' strains from Florida was predominated by E-type G (82.4%). Diversity of 'Ca. L. asiaticus' in China was also evidenced. Strains from the high altitude Yunnan Province consisted of five E-types with E-type B being the majority (62.8%), whereas strains from the low altitude coastal Guangdong Province consisted of only two E-types with E-type A as the majority (97.0%). Sequence analyses revealed that variation of DNA amplicons was due to insertion/deletion events at CLIBASIA_05650 and the downstream intergenic region.
This study demonstrated the genomic mosaicism of 'Ca. L. asiaticus' resulted from active DNA insertion/deletion activities. Analyses of strain variation depicted the significant inter- and intra-continent diversity of 'Ca. L. asiaticus'.