X

Quality Statement

Pacific Biosciences is committed to providing high-quality products that meet customer expectations and comply with regulations. We will achieve these goals by adhering to and maintaining an effective quality-management system designed to ensure product quality, performance, and safety.

X

Image Use Agreement

By downloading, copying, or making any use of the images located on this website (“Site”) you acknowledge that you have read and understand, and agree to, the terms of this Image Usage Agreement, as well as the terms provided on the Legal Notices webpage, which together govern your use of the images as provided below. If you do not agree to such terms, do not download, copy or use the images in any way, unless you have written permission signed by an authorized Pacific Biosciences representative.

Subject to the terms of this Agreement and the terms provided on the Legal Notices webpage (to the extent they do not conflict with the terms of this Agreement), you may use the images on the Site solely for (a) editorial use by press and/or industry analysts, (b) in connection with a normal, peer-reviewed, scientific publication, book or presentation, or the like. You may not alter or modify any image, in whole or in part, for any reason. You may not use any image in a manner that misrepresents the associated Pacific Biosciences product, service or technology or any associated characteristics, data, or properties thereof. You also may not use any image in a manner that denotes some representation or warranty (express, implied or statutory) from Pacific Biosciences of the product, service or technology. The rights granted by this Agreement are personal to you and are not transferable by you to another party.

You, and not Pacific Biosciences, are responsible for your use of the images. You acknowledge and agree that any misuse of the images or breach of this Agreement will cause Pacific Biosciences irreparable harm. Pacific Biosciences is either an owner or licensee of the image, and not an agent for the owner. You agree to give Pacific Biosciences a credit line as follows: "Courtesy of Pacific Biosciences of California, Inc., Menlo Park, CA, USA" and also include any other credits or acknowledgments noted by Pacific Biosciences. You must include any copyright notice originally included with the images on all copies.

IMAGES ARE PROVIDED BY Pacific Biosciences ON AN "AS-IS" BASIS. Pacific Biosciences DISCLAIMS ALL REPRESENTATIONS AND WARRANTIES, EXPRESS, IMPLIED OR STATUTORY, INCLUDING, BUT NOT LIMITED TO, NON-INFRINGEMENT, OWNERSHIP, MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. IN NO EVENT SHALL Pacific Biosciences BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, PUNITIVE, OR CONSEQUENTIAL DAMAGES OF ANY KIND WHATSOEVER WITH RESPECT TO THE IMAGES.

You agree that Pacific Biosciences may terminate your access to and use of the images located on the PacificBiosciences.com website at any time and without prior notice, if it considers you to have violated any of the terms of this Image Use Agreement. You agree to indemnify, defend and hold harmless Pacific Biosciences, its officers, directors, employees, agents, licensors, suppliers and any third party information providers to the Site from and against all losses, expenses, damages and costs, including reasonable attorneys' fees, resulting from any violation by you of the terms of this Image Use Agreement or Pacific Biosciences' termination of your access to or use of the Site. Termination will not affect Pacific Biosciences' rights or your obligations which accrued before the termination.

I have read and understand, and agree to, the Image Usage Agreement.

I disagree and would like to return to the Pacific Biosciences home page.

Pacific Biosciences
Contact:

A TAL Tale: PacBio Sequencing Helps Unravel Mechanisms of Plant Infection

Wednesday, August 19, 2020

How do bacteria manipulate plant biology to cause blight and rot? Why are some pathogen strains more virulent than others? How can we engineer resistant staple food crops? These are pressing questions facing researchers looking to sustain and increase crop production against the backdrop of a changing environment. 

For one major clade of pathogens, Xanthomonas spp, the answers lay locked within TAL effector genes (TALEs), but assembling these highly variable, repetitive regions was a long-standing obstacle. The key to finally unraveling the tangled assemblies was PacBio long-read sequencing. 

Code-breaker

Adam J. Bogdanove from Cornell University. Photo by Jesse Winter

Plant pathologist Adam J Bogdanove (@AdamBogdanove) and colleagues at Cornell University have been using SMRT Sequencing to elucidate the structure and function of TALEs and generate new insights into the mechanisms of Xanthomonas virulence.

“Repeats render TAL effector genes nearly impossible to assemble using next-generation short reads… long-read, single molecule real-time (SMRT) sequencing solves this problem,” Bogdanove wrote in one study.

Bogdanove was one of two researchers to break the TALE-DNA code in 2010 by searching for patterns in protein sequence alignments and the promoter sequences of genes upregulated by TALEs.

Drawn by David Goodsell from PDB file 3UGM.

What are TALEs?

TALEs are proteins secreted by Xanthomonas bacteria when they infect various plant species (including pepper, rice, citrus, cotton, tomato, and soybeans), causing localized leaf spot and leaf streak, or systemic black rot and leaf blight disease.

How do they work?

TALEs bind promoter sequences in the host plant and activate the expression of plant genes that aid bacterial infection. They recognize plant DNA sequences through a central repeat domain consisting of a variable number of tandem ~34 amino acid repeats that vary at only 2 positions. These two critical amino acids correspond to a DNA base in the target promoter sequence. Bogdanove figured out that HD binds to C, NI to A, NH to G, etc. 

The enabling technology

The Bogdanove lab developed a custom workflow for assembling tal-rich Xanthomonas genomes. Image: Booher, N. et al. (2015) Single molecule real-time sequencing of Xanthomonas oryzae genomes reveals a dynamic structure and complex TAL (transcription activator-like) effector gene relationships. Microbial Genomics

How did Bogdanove come to rely on SMRT Sequencing for TALE research? In a 2015 paper in Microbial Genomics, he described comparing PacBio assemblies to existing Sanger-based reference genomes of X. oryzae pathovars. The exercise revealed errors and omissions in the Sanger sequences, and the team concluded that PacBio sequencing was the best tool for generating de novo, whole-genome assemblies for Xanthomonas that accurately capture TALE genes.

Accurate assembly of TALE genes can be ensured by pre-assembling reads that contain TALE gene sequences. Together with co-author Nicholas J. Booher and others at Cornell, Bogdanove created a workflow for TALE gene assembly and prediction of their target sequences, the “pbx toolkit,” available on Github. Since then, the Bogdanove lab, along with other researchers worldwide, have used SMRT Sequencing to elucidate the molecular mechanisms of TALE function and the dance of susceptibility and resistance between pathogen and host.

Tracking virulence

A bacterial pustule caused by Xanthomonas axonopodis pv. glycines on the underside of a soybean leaf. Image: A. Robertson via Crop Protection Network

Bogdanove and colleagues have used PacBio sequencing on Xanthomonas strains that infect important food crops worldwide. He writes, “Identification of the complete sets of TALEs in different isolates can be an important first step toward development and targeted regional deployment of resistant soybean varieties, and comparison of whole genome structure across strains can yield insight into the overall genetic diversity of the pathogen.”

A 2019 Genome Biology and Evolution paper describes the complete assembly, including all TALE genes, of Xanthomonas axonopodis pv. glycines isolates collected from infected soybean plants. Surprisingly, they found that the TALEs of the three strains they sequenced were highly similar, despite having been collected over a span of 30 years and on two different continents. Bogdonove concludes that if there is “little to no genetic variation at their targets across commonly grown soybean varieties, such that there is no selective pressure on the tal genes to adapt,” these genes may be good targets for the development of resistance.

In another paper published in Frontiers in Microbiology, Bogdanove and colleagues in Iran used SMRT Sequencing to better understand the role of TALEs in the virulence of bacterial leaf streak in wheat caused by Xanthomonas translucens pv. undulosa (Xtu). 

They sequenced the genome of the highly virulent Iranian strain ICMP11055, generating a closed 4.5 Mb genome. They then compared it to the XT4699 strain from the United States, finding two major re-arrangements, nine genomic regions unique to ICMP11055, and one region unique to XT4699, as well as differences in TAL effector genes. Mutagenesis and complementation experiments indicated that at least a subset of the TALEs contribute to the virulence of these strains in wheat. 

“Our results lay the foundation for identification of important host genes activated by Xtu TALEs as targets for the development of disease resistant varieties,” the authors wrote. 

Tracking the SWEET tooth of TALEs

Finding the target genes of TALEs in plant hosts is critical to understanding the co-evolution of bacterial virulence and plant resistance.  In a Nature Communications paper, Bogdanove and his team used PacBio sequencing to link Xanthomonas citri subsp. malvacearum (Xcm) TALEs to SWEET (‘sugars will eventually be exported transporter’) target genes in cotton. By correlating cotton transcriptome profiling with Xcm TALE DNA binding site prediction, they postulated a connection between TAL effector Avrb6 and the induction of sucrose transporter GhSWEET10. 

In follow-up experiments, the authors found that “activation of GhSWEET10 by designer TAL effectors (dTALEs) restores virulence of Xcm avrb6 deletion strains, whereas silencing of GhSWEET10 compromises cotton susceptibility to infections.” “These findings advance our understanding of the disease and resistance in cotton and may facilitate the development of cotton with improved resistance to BBC.”

Blight of rice

Xanthmonas oryzae pv. Oryzae blight of rice. Image: Naqvi, S et al. (2014) Determination of antibacterial activity of various broad spectrum antibiotics against Xanthomonas oryzae pv. oryzae, a cause of bacterial leaf blight of rice. International Journal of Microbiology and Mycology

Another study of SWEET genes focused on host resistance to the rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). SWEET activation by TALEs leads to sucrose export into the xylem vessels, facilitating Xoo proliferation in rice. Researchers have identified and cultivated 42 bacterial blight resistance genes in rice, called Xa genes, which can prevent binding and activation by the cognate TALEs via two distinct mechanisms, reducing susceptibility to Xoo. All but one of the resistance genes are SWEET alleles that lack binding sites for TALEs. The final resistance gene is a mutation in a transcription factor gene that prevents TALE binding to the plant transcriptional machinery.

However, bacterial strains that can defeat these resistance mechanisms have arisen in India and Thailand. So, together with collaborators in those countries, Bogdanove sequenced the genome of one such strain from each country. While examination of the encoded TALEs revealed how the Xoo strains escaped the protective mutation of a key transcription factor gene, the mechanism for escaping protective mutations in SWEET alleles remained unclear but the data suggested an experimental path for resolving the mystery.

“The findings open a door to mechanistic understanding of the role SWEET genes play in susceptibility and illustrate the importance of complete genome sequence-based monitoring of Xoo populations in developing varieties with effective disease resistance,” the authors wrote.

Re-evaluating references

SMRT Sequencing also helped correct a long-held belief regarding Brassicaceae-infecting Xanthomonas campestris (Xc). TALE-encoding genes were thought to be absent from Xc genomes based on four reference genomic sequences. But as reported in New Phytologist, Bogdanove and colleagues from the Université de Toulouse discovered TAL genes in 26 of 49 Xc strains isolated worldwide. 

Using a combination of SMRT and TALE amplicon sequencing, they created a “TALome,” a near-complete description of the TALEs found in Xc. The new resource will “open novel perspectives for elucidating TALE-mediated susceptibility of Brassicaceae to black rot disease,” the authors wrote.

The complexity of bacterial genomes

While bacteria have a reputation for having small and tractable genomes, in truth there are many clades where the presence of numerous genes from highly repetitive gene families is common. Adding to the assembly complexity, these genes are often flanked by similarly repetitive mobile elements. PacBio sequencing offers a simple, affordable solution to closing even the most challenging bacterial genomes, enabling new insights into key biological processes. 

 

Learn more about microbial whole genome sequencing, connect with a service provider, or talk to a PacBio scientist.

 

Subscribe for blog updates:

Archives

Press Release

Pacific Biosciences Announces New Chief Financial Officer

Monday, September 14, 2020