Candidatus Liberibacter' species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from 'Candidatus Liberibacter asiaticus' (Las). In order to gain greater insight into 'Ca. Liberibacter' biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse 'Ca. Liberibacter' species, including those that can infect citrus. Our phylogenetic analysis differentiates 'Ca. Liberibacter' species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic 'Ca. Liberibacter' species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of 'Ca. Liberibacter' species, the introduction of Las in the United States and identifies promising Las targets for disease management. K E Y W O R D S 'Candidatus Liberibacter' sp., citrus greening disease, HLB, huanglongbing, phylogenomics, SEC effector | 717 THAPA eT Al.

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The citrus industry is facing an unprecedented crisis due to Huanglongbing (HLB, aka citrus greening disease), a bacterial disease associated with the pathogen Candidatus Liberibacter asiaticus (CLas) that affects all commercial varieties. Transmitted by the Asian citrus psyllid (ACP), CLas colonizes citrus phloem, leading to reduced yield and fruit quality, and eventually tree decline and death. Since adequate curative measures are not available, a key step in HLB management is to restrict the spread of the disease by identifying infected trees and removing them in a timely manner. However, uneven distribution of CLas cells in infected trees and the long latency for disease symptom development makes sampling of trees for CLas detection challenging. Here, we report that a CLas secreted protein can be used as a biomarker for detecting HLB infected citrus. Proteins secreted from CLas cells can presumably move along the phloem, beyond the site of ACP inoculation and CLas colonized plant cells, thereby increasing the chance of detecting infected trees. We generated a polyclonal antibody that effectively binds to the secreted protein and developed serological assays that can successfully detect CLas infection. This work demonstrates that antibody-based diagnosis using a CLas secreted protein as the detection marker for infected trees offers a high-throughput and economic approach that complements the approved quantitative polymerase chain reaction-based methods to enhance HLB management programs.

Citrus is one of the most economically important fruit crops in the world. Citrus psorosis is a serious disease affecting mainly oranges and mandarins in Argentina and Uruguay. The causal agent is Citrus psorosis virus (CPsV), an ophiovirus with a tripartite ssRNA genome of negative polarity. The coat protein (CP), the most abundant viral protein in infected plants, has been used to detect CPsV by TAS‐ELISA, but only biological indexing, requiring 1 year, is the current and validated technique for diagnosis of citrus psorosis. In this study, a SYBR Green RT‐qPCR protocol was developed, with primers designed to the most conserved region of the cp gene. We tested their specificity and sensitivity in comparison with TAS‐ELISA. This RT‐qPCR was applied successfully to field samples from Argentina, to a variety of isolates from different countries maintained in the greenhouse, to young seedlings and old trees from a psorosis natural transmission plot, and to transgenic citrus expressing the cp gene of CPsV or a fragment thereof. This method allowed accurate quantification of viral titer and cp gene expression in transgenic plants, which could not be detected previously. The sensitivity and reliability of quantitative CPsV detection were improved with greater speed using commercial reagents, and the sensitivity was three orders of magnitude higher than that of TAS‐ELISA. All these data encourage its validation.

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Citrus psorosis virus (CPsV) is the causal agent of psorosis, a serious and widespread citrus disease. Two syndromes of psorosis, PsA and PsB, have been described. PsB is the most aggressive and rampant form. Previously, we obtained Pineapple sweet orange plants transformed with a hairpin construct derived from the CPsV coat protein gene (ihpCP). Some of these plants were resistant to CPsV 90-1-1, a PsA isolate homologous to the transgene. In this study, we found that expression of the ihpCP transgene and siRNA production in lines ihpCP-10 and -15 were stable with time and propagation. In particular, line ihpCP-15 has been resistant for more than 2 years, even after re-inoculation. The ihpCP plants were also resistant against a heterologous CPsV isolate that causes severe PsB syndrome. Line ihpCP-15 manifested complete resistance while line ihpCP-10 was tolerant to the virus, although with variable behaviour, showing delay and attenuation in PsB symptoms. These lines are promising for a biotech product aimed at eradicating psorosis.

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‘Candidatus Liberibacter asiaticus’ (Las) is an emergent bacterial pathogen that is associated with the devastating citrus huanglongbing (HLB). Vectored by the Asian citrus psyllid, Las colonizes the phloem tissue of citrus, causing severe damage to infected trees. So far, cultivating pure Las culture in axenic media has not been successful, and dual-transcriptome analyses aiming to profile gene expression in both Las and its hosts have a low coverage of the Las genome because of the low abundance of bacterial RNA in total RNA extracts from infected tissues. Therefore, a lack of understanding of the Las transcriptome remains a significant knowledge gap. Here, we used a bacterial cell enrichment procedure and confidently determined the expression profiles of approximately 84% of the Las genes. Genes that exhibited high expression in citrus include transporters, ferritin, outer membrane porins, specific pilins, and genes involved in phage-related functions, cell wall modification, and stress responses. We also found 106 genes to be differentially expressed in citrus versus Asian citrus psyllids. Genes related to transcription or translation and resilience to host defense response were upregulated in citrus, whereas genes involved in energy generation and the flagella system were expressed to higher levels in psyllids. Finally, we determined the relative expression levels of potential Sec-dependent effectors, which are considered as key virulence factors of Las. This work advances our understanding of HLB biology and offers novel insight into the interactions of Las with its plant host and insect vector.