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 ...

Gannan Normal University

Xanthomonas citri ssp. citri (Xcc) is an important plant-pathogenic bacterium that causes citrus canker disease worldwide. PthA, a transcriptional activator-like (TAL) effector, directs the expression of the canker susceptibility gene CsLOB1. Here, we report our recent progress in the functional characterization of CsLOB1. Subcellular localization analysis of CsLOB1 protein in citrus protoplast revealed that CsLOB1 is primarily localized in the nucleus. We showed that CsLOB1 expression driven by dexamethasone (DEX) in CsLOB1-GR transgenic plants is associated with pustule formation following treatment with DEX. Pustule formation was not observed in DEX-treated wild-type plants and in non-treated CsLOB1-GR transgenic plants. Water soaking is typically associated with symptoms of citrus canker. Weaker water soaking was observed with pustule formation in CsLOB1-GR transgenic plants following DEX treatment. When CsLOB1-GR-transgenic Duncan grapefruit leaves were inoculated with Xcc306ΔpthA4 and treated with DEX, typical canker symptoms, including hypertrophy, hyperplasia and water soaking symptoms, were observed on DEX-treated transgenic plant leaves, but not on mock-treated plants. Twelve citrus genes that are induced by PthA4 are also stimulated by the DEX-induced expression of CsLOB1. As CsLOB1 acts as a transcriptional factor, we identified putative targets of CsLOB1 via bioinformatic and electrophoretic mobility shift assays. Cs2g20600, which encodes a zinc finger C3HC4-type RING finger protein, has been identified to be a direct target of CsLOB1. This study advances our understanding of the function of CsLOB1 and the molecular mechanism of how Xcc causes canker symptoms via CsLOB1.

Functional characterization of the citrus canker susceptibility gene CsLOB1.

 subsp.  () is the causal agent of citrus bacterial canker (CBC), one of the most devastating citrus diseases. Most commercial citrus varieties are susceptible to CBC. However, some citrus varieties and wild citrus germplasms are CBC resistant and are promising in genetic increases in citrus resistance against CBC. We aimed to evaluate citrus germplasms for resistance against CBC. First, we developed a rapid evaluation method based on enhanced yellow fluorescent protein (eYFP)-labeled . The results demonstrated that eYFP does not affect the growth and virulence of .  allows measurement of bacterial titers but is more efficient and rapid than the plate colony counting method. Next, we evaluated citrus germplasms collected in China. Based on symptoms and bacterial titers, we identified that two citrus germplasms ('Ichang' papeda and 'Huapi' kumquat) are resistant, whereas eight citrus germplasms ('Chongyi' wild mandarin, 'Mangshan' wild mandarin, 'Ledong' kumquat, 'Dali' citron, 'Yiliang' citron, 'Longyan' kumquat, 'Bawang' kumquat, and 'Daoxian' wild mandarin) are tolerant. In summary, we have developed a rapid evaluation method to test the resistance of citrus plants against CBC. This method was successfully used to identify two highly canker-resistant citrus germplasms and eight citrus germplasms with canker tolerance. These results could be leveraged in traditional breeding contexts or be used to identify canker resistance genes to increase the disease resistance of commercial citrus varieties via biotechnological approaches.

Rapid Evaluation of the Resistance of Citrus Germplasms Against  subsp. .

 subsp.  () is the agent of citrus bacterial canker (CBC) disease, which has significantly reduced citrus quantity and quality in many producing areas worldwide. Copper-based bactericides are the primary products for CBC control and management, but the problems derived from copper-resistant and environmental contamination have become issues of anxiety. Thus, there is a need to find alternative antibacterial products instead of relying on a single type of agent. This study developed a method to evaluate the inhibition of antibacterial agents using the fluorescence-labeled recombinant  strain (). The optimization of timelines and parameters for the evaluation of antibacterial agents involved the use of a Spark™ multimode microplate reader. This evaluation and screening method can be applied to bactericides, cocktail-mixture formulations, antagonistic bacteria, and derived metabolites. The results showed that the minimum inhibitory concentration (MIC) of commercial bactericides determined by fluorescence agrees with the MIC values determined by the conventional method. A screened cocktail-mixture bactericide presents more activity than the individual agents during the protective effects. Notably, this method has been further developed in the screening of -antagonistic bacterial strains. In summary, we provide a validated strategy for screening and evaluation of different antibacterial components for inhibition against  for CBC control and management.

A Fluorescent Reporter-Based Evaluation Assay for Antibacterial Components Against  subsp. .

Histone methylation, which is critical for transcriptional regulation and various biological processes in eukaryotes, is a reversible dynamic process regulated by histone methyltransferases (HMTs) and histone demethylases (HDMs). This study determined the function of 5 HMTs (, , , , and ) and 1 HDMs () in the phytopathogenic fungus  by analyzing targeted gene deletion mutants. The vegetative growth, conidiation, and pathogenicity of ∆ and ∆ were severely inhibited indicating that  and  play critical roles in cell development in . Multiple stresses analysis revealed that both  and  were involved in the adaptation to cell wall interference agents and osmotic stress. Meanwhile, ∆ and ∆ displayed serious vegetative growth defects in sole carbon source medium, indicating that  and  play an important role in carbon source utilization. In addition, ∆ colony displayed white in color, while the wild-type colony was dark brown, indicating  is an essential gene for melanin biosynthesis in .  was required for the resistance to oxidative stress. On the other hand, all of ∆, ∆, and ∆ mutants displayed wild-type phenotype in vegetative growth, multi-stress resistance, pathogenicity, carbon source utilization, and melanin biosynthesis. To explore the regulatory mechanism of  and , RNA-seq of these mutants and wild-type strain was performed. Phenotypes mentioned above correlated well with the differentially expressed genes in ∆ and ∆ according to the KEGG and GO enrichment results. Overall, our study provides genetic evidence that defines the central role of HMTs and HDMs in the pathological and biological functions of .

Histone Methylation Is Required for Virulence, Conidiation, and Multi-Stress Resistance of .

The plant hormone salicylic acid (SA) receptor NONEXPRESSOR OF PATHOGENESIS-RELATED PROTEINS1 (NPR1) plays a critical role for plant defense against biotrophic and hemi-biotrophic pathogens. In a milestone paper, Kumar, Zavaliev, Wu et al. unraveled the structural basis for the assembly of an enhanceosome by NPR1 in activating the expression of plant defense genes.

A vigilant gliding bird protects plants.

Citrus is an essential horticultural fruit whose yield and quality are affected by salinity all over the world. The recognition and adaptive regulation of citrus against salt stress are important areas for cultivar improvement, but the vascular system signal transduction mechanism of the plant response to salt stress remains elusive. In this study, we constructed a dodder () linked Hamlin sweet orange () plant community in which deliver a vascular signal through the dodder in response to salt stress. RNA-seq technology was used to analyze the gene expression profile of citrus leaves after salt treatment. The results showed that a vascular signal was transmitted to a dodder-linked host plant, triggering a transcriptional response to salt stress. However, the phenotypic and transudative ability of the dodder changed after 24 h. The salt treatment group (Group S) and the dodder-linked group (Group D) respectively contained 1,472 and 557 differentially expressed genes (DEGs). 454 of which were common to both groups. The results of our analysis revealed that the gene expression categories in Group D represented a highly consistent trend compared to the group S plants, indicating that the dodder-bridged vascular signals activated the stress-response of citrus leaves for transcriptomic reconfiguration. The KEGG pathway database and an analysis of key drivers revealed that phenylpropanoid biosynthesis, photosynthesis-antenna proteins, starch and sucrose metabolism, plant hormone signal transduction, circadian rhythm, and MAPK signaling pathways were significantly enriched as the critical genes during salt stress. A systemic signal in the dodder-bridged host significantly regulated abiotic stress-related secondary metabolic pathways, including those for phenylpropanoids, lignin, and lignans. The physiological indexes of photosynthetic intensity, respiration, and attractiveness among communities supported the transcriptional changes. Thus, our results indicate that salt stress-induced vascular system signals can be transmitted through the vascular system of a dodder linking citrus plants, revealing the genetic regulation and physiological changes of citrus leaves responding to plant stress signal transmission.

Dodder-transmitted mobile systemic signals activate a salt-stress response characterized by a transcriptome change in .

Highly efficient genetic transformation technology is greatly beneficial for crop gene function analysis and precision breeding. However, the most commonly used genetic transformation technology for woody plants, mediated by , is time-consuming and inefficient, which limits its utility for gene function analysis. In this study, a simple, universal, and highly efficient genetic transformation technology mediated by  K599 is described. This technology can be applied to multiple citrus genotypes, and only 2-8 weeks were required for the entire workflow. Genome-editing experiments were simultaneously conducted using 11 plasmids targeting different genomic positions and all corresponding transformants with the target knocked out were obtained, indicating that -mediated genome editing was highly efficient. In addition, the technology is advantageous for investigation of specific genes (such as ) for obtaining "hard-to-get" transgenic root tissue. Furthermore,  can be used for direct viral vector inoculation on citrus bypassing the requirement for virion enrichment in tobacco, which facilitates virus-induced gene silencing and virus-mediated gene expression. In summary, we established a highly efficient genetic transformation technology bypassing tissue culture in citrus that can be used for genome editing, gene overexpression, and virus-mediated gene function analysis. We anticipate that by reducing the cost, required workload, experimental period, and other technical obstacles, this genetic transformation technology will be a valuable tool for routine investigation of endogenous and exogenous genes in citrus.

Shuo Duan

China-USA Citrus Huanglongbing Joint Laboratory,
National Navel Orange Engineering Research Center,
China-USA Citrus Huanglongbing Joint Laboratory, National Navel Orange Engineering Research Center

Gannan Normal University

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

Shuo Duan

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Gannan Normal University

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

China-USA Citrus Huanglongbing Joint Laboratory,
National Navel Orange Engineering Research Center,
China-USA Citrus Huanglongbing Joint Laboratory, National Navel Orange Engineering Research Center