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The citrus industry is facing an unprecedented challenge from Huanglongbing (HLB). All cultivars can be affected by the HLB-associated bacterium ‘Candidatus Liberibacter asiaticus’ ( C Las) and there is no known resistance. Insight into HLB pathogenesis is urgently needed in order to develop effective management strategies. Here, we use Sec-delivered effector 1 (SDE1), which is conserved in all C Las isolates, as a molecular probe to understand C Las virulence. We show that SDE1 directly interacts with citrus papain-like cysteine proteases (PLCPs) and inhibits protease activity. PLCPs are defense-inducible and exhibit increased protein accumulation in C Las-infected trees, suggesting a role in citrus defense responses. We analyzed PLCP activity in field samples, revealing specific members that increase in abundance but remain unchanged in activity during infection. SDE1 -expressing transgenic citrus also exhibit reduced PLCP activity. These data demonstrate that SDE1 inhibits citrus PLCPs, which are immune-related proteases that enhance defense responses in plants. Greening disease threatens the productivity of citrus crops worldwide yet the pathosystem is poorly understood. Here, Clark et al. show that an effector cloned from the associated bacteria can suppress host plant papain-like cysteine proteases' activity, suggesting its probable role in pathogenesis.

Huanglongbing (HLB) is a bacterial infection of citrus trees transmitted by the Asian citrus psyllid Diaphorina citri . Mitigation of HLB has focused on spraying of insecticides to reduce the psyllid population and removal of trees when they first show symptoms of the disease. These interventions have been only marginally effective, because symptoms of HLB do not appear on leaves for months to years after initial infection. Limited knowledge about disease spread during the asymptomatic phase is exemplified by the heretofore unknown length of time from initial infection of newly developing cluster of young leaves, called flush, by adult psyllids until the flush become infectious. We present experimental evidence showing that young flush become infectious within 15 d after receiving an inoculum of Candidatus Liberibacter asiaticus (bacteria). Using this critical fact, we specify a microsimulation model of asymptomatic disease spread and intensity in a grove of citrus trees. We apply a range of psyllid introduction scenarios to show that entire groves can become infected with up to 12,000 psyllids per tree in less than 1 y, before most of the trees show any symptoms. We also show that intervention strategies that reduce the psyllid population by 75% during the flushing periods can delay infection of a full grove, and thereby reduce the amount of insecticide used throughout a year. This result implies that psyllid surveillance and control, using a variety of recently available technologies, should be used from the initial detection of invasion and throughout the asymptomatic period. Significance Huanglongbing (HLB) is a vector-transmitted bacterial infection of citrus trees that poses a major threat to the citrus industry in Florida, Texas, and California. Current control strategies that focus on the vector, the Asian citrus psyllid Diaphorina citri , are usually initiated when the trees become symptomatic, anywhere from 10 mo to several years after initial infection. We show, experimentally, that newly infected young leaves can become infectious within 10–15 d after receiving an inoculum of bacteria from an adult psyllid. We then show by microsimulation of the asymptomatic spread of HLB through a grove under different invasion scenarios and control strategies that reduction of up to 75% of adult psyllids and nymphs can enhance citrus production.

Candidatus Liberibacter asiaticus ( C Las), a bacterium transmitted by the Asian citrus psyllid, Diaphorina citri , is the causal agent of citrus greening disease, or Huanglongbng (HLB). Currently, vector population suppression with insecticides and tree removal are the most effective strategies for managing the HLB pathosystem. In this study, we assessed the bactericidal capabilities of 2′-deoxy-2′-fluoro- d -arabinonucleic acid antisense oligonucleotides (FANA ASO) both in vitro and in vivo by (1) confirming their capacity to penetrate insect cells, (2) silencing bacterial essential genes, and (3) quantifying reductions in bacterial titer and D. citri transmission. We confirmed that FANA ASO are able to penetrate insect cells without the use of a delivery agent. Expression of an essential gene in the D. citri endosymbiont, Wolbachia (wDi), significantly decreased by 30% following incubation with a wDi-specific FANA ASO. Viability of isolated wDi cells also decreased in response to the FANA ASO treatment. Delivery of a C Las-specific FANA ASO to infected adult D. citri in feeding assays resulted in significant silencing of a C Las essential gene. C Las relative density and transmission were significantly lower among D. citri fed FANA ASO in diet compared to untreated insects. Root infusions of a C Las-specific FANA ASO into infected Citrus trees significantly reduced C Las titer during a 30-day trial. Our results suggest that FANA ASO targeting insect-transmitted plant bacteria or insect endosymbionts may be useful tool for integrated management of agricultural pathogens.

Citrus huanglongbing (HLB), caused by “Candidatus Liberibacter” spp., is one of the most disastrous citrus diseases worldwide. HLB‐affected citrus fruits are significantly more acidic than healthy fruits. However, the molecular mechanism behind this phenomenon remains to be elucidated. Here, we report that HLB‐affected fruits have higher levels of citric acid (CA) than healthy fruits. Moreover, Citrus PH4 (CitPH4), which encodes a MYB transcription factor that functions as a key regulator of CA accumulation, was upregulated in HLB‐affected fruits relative to healthy fruits. Heterologous overexpression of CitPH4 in tobacco (Nicotiana tabacum) plants enhanced tolerance to HLB. Subsequently, overexpression and gene‐editing experiments indicated that CitPH4 can affect the salicylic acid (SA) pathway, which directly binds to and activates the promoter of CsPBS3, a key gene of SA biosynthesis. HLB‐affected fruits had higher SA levels than healthy fruits. Furthermore, application of SA activated CA biosynthesis and application of CA activated SA biosynthesis and signalling in citrus fruits and decreased “Candidatus Liberibacter asiaticus” (CLas) titres in infected leaves. This work suggests that CitPH4 is a key node between CA and SA, thus revealing crosstalk between defence responses and fruit quality in citrus. The transcription factor CitPH4 is induced by “Candidatus Liberibacter asiaticus” infection and reciprocally activates salicylic acid and citric acid biosynthesis in citrus fruits as citrus immune response.

Citrus huanglongbing (HLB) disease is caused by endogenous, phloem-restricted, Gram negative, uncultured bacteria named Candidatus Liberibacter africanus (CaLaf), Ca. L. asiaticus (CaLas), and Ca. L. americanus (CaLam), depending on the continent where the bacteria were first detected. The Asian citrus psyllid vector, Diaphorina citri, transmits CaLas and CaLam and both Liberibacter species are present in Brazil. Several studies of the transcriptional response of citrus plants manifesting HLB symptoms have been reported, but only for CaLas infection. This study evaluated the transcriptional reprogramming of a susceptible genotype of sweet orange challenged with CaLam, using a customized 385K microarray containing approximately 32,000 unigene transcripts. We analyzed global changes in gene expression of CaLam-infected leaves of sweet orange during the symptomatic stage of infection and compared the results with previously published microarray studies that used CaLas-infected plants. Twenty candidate genes were selected to validate the expression profiles in symptomatic and asymptomatic PCR-positive leaves infected with CaLas or CaLam. The microarray analysis identified 633 differentially expressed genes during the symptomatic stage of CaLam infection. Among them, 418 (66%) were upregulated and 215 (34%) were down regulated. Five hundred and fourteen genes (81%) were orthologs of genes from Arabidopsis thaliana. Gene set enrichment analysis (GSEA) revealed that several of the transcripts encoded transporters associated with the endomembrane system, especially zinc transport. Among the most biologically relevant gene transcripts in GSEA were those related to signaling, metabolism and/or stimulus to hormones, genes responding to stress and pathogenesis, biosynthesis of secondary metabolites, oxidative stress and transcription factors belonging to different families. Real time PCR of 20 candidate genes validated the expression pattern of some genes in symptomatic and asymptomatic leaves infected with CaLam or CaLas. Many gene transcripts and biological processes are significantly altered upon CaLam infection. Some of them had been identified in response to CaLas infection, while others had not been previously reported. These data will be useful for selecting target genes for genetic engineering to control HLB.

Huanglongbing (HLB) is the most destructive disease that affects citrus worldwide. The disease has been associated with Candidatus Liberibacter. HLB diseased citrus plants develop a multitude of symptoms including zinc and copper deficiencies, blotchy mottle, corky veins, stunting, and twig dieback. Ca. L. asiaticus infection also seriously affects the roots. Previous study focused on gene expression of leaves and fruit to Ca. L. asiaticus infection. In this study, we compared the gene expression levels of stems and roots of healthy plants with those in Ca. L. asiaticus infected plants using microarrays. Affymetrix microarray analysis showed a total of 988 genes were significantly altered in expression, of which 885 were in the stems, and 111 in the roots. Of these, 551 and 56 were up-regulated, while 334 and 55 were down-regulated in the stem and root samples of HLB diseased trees compared to healthy plants, respectively. Dramatic differences in the transcriptional responses were observed between citrus stems and roots to Ca. L. asiaticus infection, with only 8 genes affected in both the roots and stems. The affected genes are involved in diverse cellular functions, including carbohydrate metabolism, cell wall biogenesis, biotic and abiotic stress responses, signaling and transcriptional factors, transportation, cell organization, protein modification and degradation, development, hormone signaling, metal handling, and redox. Microscopy analysis showed the depletion of starch in the roots of the infected plants but not in healthy plants. Collapse and thickening of cell walls were observed in HLB affected roots, but not as severe as in the stems. This study provides insight into the host response of the stems and roots to Ca. L. asiaticus infection.

The Asian citrus psyllid ( Diaphorina citri ) transmits the bacterium ‘ Candidatus Liberibacter asiaticus’ (CLas), which causes huanglongbing (citrus greening) disease, in a circulative-propagative manner. We compared CLas inoculation efficiency of D. citri nymphs and adults into healthy (uninfected) citron leaves when both vector stages were reared from eggs on infected plants. The proportion of CLas-positive leaves was 2.5% for nymphs and 36.3% for adults. CLas acquisition by early instar nymphs followed by dissections of adults and 4th instar nymphs revealed that CLas bacterium had moved into the head-thorax section (containing the salivary glands) in 26.7–30.0% of nymphs and 37–45% of adults. Mean Ct values in these sections were 31.6–32.9 and 26.8–27.0 for nymphs and adults, respectively. Therefore, CLas incidence and titer were higher in the head-thorax of adults than in nymphs. Our results suggest that following acquisition of CLas by early instar D. citri nymphs, emerging adults inoculate the bacteria into citrus more efficiently than nymphs because adults are afforded a longer latent period necessary for multiplication and/or translocation of CLas into the salivary glands of the vector. We propose that CLas uses D. citri nymphs mainly for pathogen acquisition and multiplication, and their adults mainly for pathogen inoculation and spread.

\"Candidatus Liberibacter asiaticus\" (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the \"Ca. Liberibacter\" genera.

\"Candidatus Liberibacter solanacearum\" (Lso) has emerged as a serious threat world-wide. Five Lso haplotypes have been identified so far. Haplotypes A and B are present in the Americas and/or New Zealand, where they are vectored to solanaceous plants by the potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae). The fastidious nature of these pathogens has hindered the study of the interactions with their eukaryotic hosts (vector and plant). To understand the strategies used by these pathogens to infect their vector, the effects of each Lso haplotype (A or B) on psyllid fitness was investigated, and genome-wide transcriptomic and RT-qPCR analyses were performed to evaluate Lso gene expression in association with its vector. Results showed that psyllids infected with haplotype B had significantly lower percentage of nymphal survival compared to psyllids infected with haplotype A. Although overall gene expression across Lso genome was similar between the two Lso haplotypes, differences in the expression of key candidate genes were found. Among the 16 putative type IV effector genes tested, four of them were differentially expressed between Lso haplotypes, while no differences in gene expression were measured by qPCR or transcriptomic analysis for the rest of the genes. This study provides new information regarding the pathogenesis of Lso haplotypes in their insect vector.

The Liberibacter genus comprises insect endosymbiont bacterial species that cause destructive plant diseases, including Huanglongbing in citrus and zebra chip in potato. To date, pathogenic ‘ Candidatus Liberibacter spp.’ (CLs) remain uncultured, therefore the plant-associated Liberibacter crescens (Lcr), only cultured species of the genus, has been used as a biological model for in vitro studies. Biofilm formation by CLs has been observed on the outer midgut surface of insect vectors, but not in planta. However, the role of biofilm formation in the life cycle of these pathogens remains unclear. Here, a model system for studying CLs biofilms was developed using Lcr. By culture media modifications, bovine serum albumin (BSA) was identified as blocking initial cell-surface adhesion. Removal of BSA allowed for the first time observation of Lcr biofilms. After media optimization for biofilm formation, we demonstrated that Lcr attaches to surfaces, and form cell aggregates embedded in a polysaccharide matrix both in batch cultures and under flow conditions in microfluidic chambers. Biofilm structures may represent excellent adaptive advantages for CLs during insect vector colonization helping with host retention, immune system evasion, and transmission. Future studies using the Lcr model established here will help in the understanding of the biology of CLs.