Field-Deployable <em>Candidatus</em> Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

The early detection of Candidatus Liberibacter asiaticus (CLas) by citrus growers facilitates early intervention and prevents the spread of disease. A ...

University of Florida - Institute of Food and Agricultural Sciences

Citrus canker, caused by Xanthomonas citri subsp. citri, is an important citrus disease that causes significant economic losses worldwide. All commercial citrus varieties are susceptible to citrus canker. Currently, chemical control with copper based products is the main approach to control X. citri subsp. citri dispersal and plant colonization. However, extensive use of copper compounds can result in copper-resistant strains and cause adverse effects on the environment. Alternatives to chemical control involve the activation of citrus immunity to control the disease. Here, we investigated the ability of multiple rhizobacteria to induce a systemic defense response in cultivar Duncan grapefruit. Burkholderia territorii strain A63, Burkholderia metallica strain A53, and Pseudomonas geniculata strain 95 were found to effectively activate plant defense and significantly reduce symptom development in leaves challenged with X. citri subsp. citri. In the priming phase, root application of P. geniculata induced the expression of salicylic acid (SA)-signaling pathway marker genes (PR1, PR2, PR5, and salicylic acid carboxyl methyltransferase [SAM-SACM]). Gene expression analyses after X. citri subsp. citri challenge showed that root inoculation with P. geniculata strain 95 increased the relative levels of phenylalanine ammonia lyase 1 and SAM-SACM, two genes involved in the phenylpropanoid pathway as well as the biosynthesis of SA and methyl salicylate (MeSA), respectively. However, hormone analyses by UPLC-MS/MS showed no significant difference between SA in P. geniculata-treated plants and control plants at 8 days post-beneficial bacteria root inoculation. Moreover, P. geniculata root-treated plants contained higher reactive oxygen species levels in aerial tissues than control plants 8 days post-treatment application. This study demonstrates that rhizobacteria can modulate citrus immunity resulting in a systemic defense response against X. citri subsp. citri under greenhouse conditions.

Induced Systemic Resistance Against Citrus Canker Disease by Rhizobacteria.

Genome sequencing is commonly used in research laboratories right now thanks to the rise of high-throughput sequencing with higher speed and output-to-cost ratios. Here, we summarized the application of genomics in different aspects of plant bacterial pathosystems. Genomics has been used in studying the mechanisms of plant-bacteria interactions, and host specificity. It also helps with taxonomy, study of non-cultured bacteria, identification of causal agent, single cell sequencing, population genetics, and meta-transcriptomic. Overall, genomics has significantly improved our understanding of plant-microbe interaction.

Where are we going with genomics in plant pathogenic bacteria?

Citrus huanglongbing (HLB) is one of the most destructive diseases affecting citrus plants. ' Liberibacter asiaticus', an uncultivated α-proteobacteria, is the most widely spread causal agent of HLB and is transmitted by the Asian citrus psyllid . '. L. asiaticus' attachment to the psyllid midgut is believed to be critical to further infect other organs, including the salivary gland. In this study, the type IVc tight adherence (Tad) pilus locus encoded by ' L. asiaticus' was characterized. The Tad loci are conserved among members of Rhizobiaceae, including ' L. asiaticus' and  spp. Ectopic expression of the ' L. asiaticus'  gene, an ATPase essential for the biogenesis and secretion of the Tad pilus, restored the adherence phenotype in  mutant of , indicating CpaF of ' L. asiaticus' was functional and critical for bacterial adherence mediated by Tad pilus. Quantitative reverse transcription PCR (qRT-PCR) analysis revealed that ' L. asiaticus' Tad pilus-encoding genes and ' L. asiaticus' pilin gene  were upregulated in psyllids compared with in planta. A bacterial one-hybrid assay showed that ' L. asiaticus' VisN and VisR, members of the LuxR transcriptional factor family, were bound to the  promoter. VisNR regulate . Negative regulation of the  promoter by both VisN and VisR was demonstrated using a shuttle strategy, with analysis of the phenotypes and immunoblotting together with quantification of the expression of the  promoter fused to the β-galactosidase reporter gene. Comparative expression analysis confirmed that ' L. asiaticus'  was less expressed in the psyllid than in the plant host. Further, motility and biofilm phenotypes of the  mutant of  were fully complemented by expressing ' L. asiaticus'  together. The physical interaction between VisN and VisR was confirmed by pull-down and stability assays. The interaction of the  promoter with VisR was verified by electrophoretic mobility shift assay. Taken together, the results revealed the contribution of the Tad pilus apparatus in the colonization of the insect vector by ' L. asiaticus' and shed light on the involvement of VisNR in regulation of the Tad locus.

The Tad Pilus Apparatus of ' Liberibacter asiaticus' and Its Regulation by VisNR.

The Citrus Huanglongbing Crisis and Potential Solutions.

Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. 'Candidatus Liberibacter asiaticus' (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin-encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin-encoding genes. Only flgJ but not flgJ restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull-down assays demonstrated that FlgJ interacts with FlgB but not with FliE. Ectopic expression of flaA in A. tumefaciens mutants restored the swimming motility of ∆flaA mutant and ∆flaAD mutant, but not that of the null mutant ∆flaABCD. No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta. In addition, western blotting using flagellin-specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta. The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

The flagella of 'Candidatus Liberibacter asiaticus' and its movement in planta.

Citrus Huanglongbing (HLB), caused by  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 () suppresses the hypersensitive response induced by  ssp.  () and reduces the expression of immunity-related genes. SDE15 also suppresses the hypersensitive response triggered by the  effector protein AvrBsT in , suggesting that it may be a broad-spectrum suppressor of plant immunity. SDE15 interacts with the citrus protein CsACD2, a homolog of Arabidopsis () ACCELERATED CELL DEATH 2 (ACD2). SDE15 suppression of plant immunity is dependent on , and overexpression of  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.

Nian Wang

Citrus Research and Education Center,
Department of Microbiology and Cell Science,
Citrus Research and Education Center, Department of Microbiology and Cell Science

University of Florida - Institute of Food and Agricultural Sciences

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Nian Wang

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Citrus Research and Education Center,Department of Microbiology and Cell Science,Citrus Research and Education Center, Department of Microbiology and Cell Science

University of Florida - Institute of Food and Agricultural Sciences

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a

Citrus Research and Education Center,
Department of Microbiology and Cell Science,
Citrus Research and Education Center, Department of Microbiology and Cell Science