Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp

Abstract

A major bottleneck in identifying therapies to control citrus greening and other devastating plant diseases caused by fastidious pathogens is our inability to culture the pathogens in defined media or axenic cultures. As such, conventional approaches for antimicrobial evaluation (genetic or chemical) rely on time-consuming, low-throughput and inherently variable whole-plant assays. Here, we report that plant hairy roots support the growth of fastidious pathogens like Candidatus Liberibacter spp., the presumptive causal agents of citrus greening, potato zebra chip and tomato vein greening diseases. Importantly, we leverage the microbial hairy roots for rapid, reproducible efficacy screening of multiple therapies. We identify six antimicrobial peptides, two plant immune regulators and eight chemicals which inhibit Candidatus Liberibacter spp. in plant tissues. The antimicrobials, either singly or in combination, can be used as near- and long-term therapies to control citrus greening, potato zebra chip and tomato vein greening diseases.

Fig. 1. Candidatus Liberibacter solanacearum and Candidatus Liberibacter asiaticus in potato and citrus hairy roots.

a, b Hairy roots (indicated by arrows) from Candidatus Liberibacter solanacearum (CLso)-infected potato (a) and Candidatus Liberibacter asiaticus (CLas)-infected citrus explants (b). c, d Visual confirmation of GFP expression in hairy roots by fluorescence microscopy. Scale bars, 1 cm. e, f Detection of CLso and CLas in the hairy roots by PCR amplification of diagnostic markers specific to CLso (16S rDNA) and CLas (rplk04/J5 and RNR). GFP, rolB, and rolC encoded on the Ti and Ri plasmids, respectively, and co-transformed into the hairy roots, were used as additional markers for hairy root authenticity; RPL2 and GAPC2 are endogenous potato and citrus genes, respectively, used as genomic DNA controls for PCR. “L” and “HR” indicate leaf and hairy root samples, respectively. “+” indicates positive controls used for the respective PCR amplifications. g, h Temporal growth curves of CLso and CLas in the hairy roots. The Ct values of CLso and CLas in hairy root samples collected at different days in propagation are plotted, whereas the approximate genome equivalents (GE) per nanogram of root genomic DNA are indicated above the bar graph columns. Error bars represent ± standard error of mean (n = 4 and n = 3 for g and h, respectively). Uncropped raw agarose gel images used to prepare e and f are presented in Supplementary Fig. 10. Source data underlying Fig. 1g, h are provided as a Source Data file.

Fig. 2. Screening and identification of antimicrobial genes that confer tolerance to Candidatus Liberibacter spp.

a Schematic of a typical transgene overexpression construct with a gene and a GFP marker cassette (included to serve as a visual marker for construct integration in the hairy roots). b Representative images of healthy and CLso-potato hairy roots at 30 days post transformation (i.e., 30 dpt) with control (GFP alone) or with putative disease-resistance genes from Arabidopsis (AtNPR1) and tomato (SlNPR1). c Visualization of the hairy roots under a fluorescence microscope. Scale bars, 1 cm. d Quantification of relative CLso titers in transformed hairy roots at 30 dpt, compared to control (GFP alone). Error bars represent ± standard error of mean (n = 3). p Values were calculated by two-sample t test (one-tailed). The experiment was independently repeated two times, and all attempts of replication were successful. e Relative expression of PR1-like and PR3-like and WRKY6-like genes in AtNPR1- and SlNPR1-expressing hairy roots. Error bars represent ± standard error of mean (n = 3). p Values were calculated by two-sample t test (one-tailed) relative to respective control samples. f Quantification of salicylic acid (SA) in the transformed hairy roots showed lower SA accumulation in AtNPR1 and SlNPR1 overexpressors. Error bars represent ± standard error of mean (n = 4). p Values were calculated by two-sample t test (one-tailed). g Evaluation of eight putative antimicrobial peptides in CLso-potato and CLas-citrus hairy roots. Relative CLso and CLas titers were calculated from five biological replicates. Error bars represent ± standard error of mean (n = 5). p Values were calculated by two-sample t test (one-tailed) relative to infected controls. The experiment was independently repeated two times, and all attempts of replication were successful. Source data underlying Fig. 2d–f are provided as a Source Data file.

Fig. 3. Evaluation of genome editing in microbial hairy roots.

a Typical CRISPR–Cas9 gene editing construct. The underlined sequence is the target DNA site used in the sgRNA. b–d Transformation of Cas9 alone (control) and Cas9–sgGFP targeting stably expressed GFP transgene in healthy and Candidatus Liberibacter solanacearum (CLso) hairy roots. The loss of GFP fluorescence in Cas9–sgGFP, but not in Cas9 alone, indicates successful editing of the GFP transgene. Scale bars, 1 cm. The experiment was independently repeated two times, and all attempts of replication were successful. e, f Amplicon sequencing confirmed gene editing in the target site (GFP), as indicated by presence of indels in the Cas9–sgGFP hairy roots, but not in Cas9-alone hairy roots. Frequencies of indels detected were ~86 and 100% in the healthy and CLso hairy roots, respectively. Uncropped raw agarose gel images used to prepare Supplementary Fig. 3b are presented in Supplementary Fig. 11.

Fig. 4. High-throughput screening and identification of small molecules that confer tolerance to Candidatus Liberibacter spp.

a–c Multi-well microbial hairy root culture plates were used to conduct high-throughput screening of ~220 compounds (Supplementary Dataset 1). The efficacy data for nine selected hit compounds were re-assayed at 10 and 25 µM, respectively, in a, b CLso and a, c CLas hairy roots. Untreated (UT) and tetracycline (TC)-treated hairy roots (at 250 and 500 ppm, respectively, for the CLso and CLas assays) were used as positive and negative controls. The bacterial titers were estimated by qPCR after 72 h of treatment with each compound and plotted relative to those of untreated samples (set to 100%). Error bars represent ± standard error of mean (n = 5). p Values were calculated by two-sample t test (one-tailed) relative to untreated samples. d–m Chemical structures of d tetracycline and the nine hits: e aminocaproic acid (#1), f carbinoxamine maleate (#2), g chloroxylenol (#3), h chlorpropamide (#4), i chlortetracycline (#5), j cinoxacin (#6), k cortisone acetate (#7), l duartin (#8), and m cyclopentolate hydrochloride (#9). The structures of the different chemical compounds were retrieved from the ChemSpider database (http://www.chemspider.com/) (last accessed on 15 October 2020). Source data underlying Fig. 4b, c are provided as a Source Data file.

Fig. 5. In planta application of small molecules confer tolerance to Candidatus Liberibacter spp. in potatoes.

a Visual symptoms of plants at 28 days post infection (dpi). Tetracycline (TC, 250 ppm) and three selected small molecules (#3, #8, and #9; see Fig. 4) were sprayed on the plants at two concentrations (10 and 25 µM) as indicated. Disease symptoms of chlorosis, necrosis, leaf curling, and wilting were monitored. b CLso titers were determined by qPCR at 28 dpi. All titers are plotted relative to those of untreated samples, which were set to 100%. Error bars represent ± standard error of mean (n = 4). p Values were calculated by two-sample t test (one-tailed) relative to untreated samples. Source data underlying Fig. 5b are provided as a Source Data file.