Eliza Po-Iian Loo

Bacterial leaf blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), causes yield losses exceeding 50% in affected areas, including the Bagré rice plain in Burkina Faso. Genome-edited (GE’d) rice lines have been successful in tackling BB. Modifications in the Xoo virulence protein target site upstream of three SWEET susceptibility genes in two elite rice varieties, IR64 and Ciherang-Sub1, have been demonstrated to confer broad-spectrum resistance to Asian and East African Xoo strains. Here, we evaluate the potential of the GE’d lines as a solution for BB management in Burkina Faso. We challenged the GE’d lines against five locally collected Burkinabè Xoo strains under controlled greenhouse conditions and assessed their agro-morphological performance under field conditions representative of local agroecological conditions. Greenhouse pathogen assays demonstrated that GE’d IR64 and Ciherang-Sub1 lines were resistant to all tested local Xoo strains across three successive generations. We identified TalC as the primary disease-causing effector in the local Xoo populations. Irrigated field trials conducted over two seasons in the Kou Valley, Burkina Faso, revealed absence of agro-morphological penalties in GE’d lines compared to their parental wild-type lines. Observed trait variation was attributable to environmental fluctuations rather than genomic modifications. Collectively, our findings demonstrate that genome editing of the rice lines does not impose growth penalties, and support the suitability of GE’d IR64 and Ciherang-Sub1 for large-scale adoption in Burkina Faso, pending multi-location validation and introgression into locally adapted varieties.

SUMMARY: Controlled plant growth in laboratories can be achieved by cultivating plants under sterile or axenic conditions on predefined synthetic growth media, typically supplemented with sugar. In nature, plants do not receive exogenous sugar supplies, form symbiosis with microbes, and plant growth is influenced by soil edaphic factors. Thus, physiological and multi‐omic analyses of plants grown on synthetic media will differ from those of soil‐grown plants due to the influence of sucrose, and the absence of microbiota and soil edaphic factors on plant growth. The rapid advances in spatial omics call for accurate characterization of plants grown under conditions similar to soil. To address the issue, we developed Artificial Soil (ArtSoil), a growth medium containing essential nutrients for plant growth, and aqueous soil extract (ASE) to maintain soil microbiomes and edaphic factors, simultaneously eliminating the need for sugar supplementation in the medium. We compared Arabidopsis thaliana grown on conventional media and on ArtSoil under various growth conditions. We showed that complex soil microbiota in ArtSoil promote plant growth without physiological side effects induced by sucrose. We demonstrate an application for ArtSoil in single‐cell transcriptomics and report microbiota‐induced cell‐type‐specificity in immune and nitrogen signaling. We tested ArtSoil with six types of ASEs to demonstrate its potential to decouple nutrient effects from microbiota in plant growth. We conclude that ArtSoil offers a more physiologically relevant alternative to conventional media for studying plant growth within a soil‐like context.

The virulence of Xanthomonas oryzae pv. oryzae, the causal agent of bacterial blight (BB) of rice, critically depends on the activation of SWEET sucrose uniporters of the host. To date the role of SWEET-released sucrose for virulence remains unclear. We here identified the sux locus of Xoo consisting of a LacI-type repressor (SuxR), an outer membrane TonB-like porin (SuxA), an inner membrane MFS H+-symporter (SuxC), and a cytosolic sucrose hydrolase (SuxB). Structural and functional analyses demonstrate that SuxB has exclusive sucrose hydrolase activity. Mutant analyses show that the transporter SuxC and the sucrose hydrolase are necessary for growth of bacteria on sucrose, while SuxA is not essential, likely due to the ability of other porins to transport sucrose across the outer membrane. Consistent with a role of SuxR as a sucrose repressor, transcriptome studies show sucrose-dependent regulation of the suxA/suxB genes. Besides a role of sucrose for reproduction, we found that sucrose promotes motility, EPS production, biofilm formation, and virulence. Notably, the SuxC sucrose H+-symporter and the sucrose hydrolase SuxB were required for full virulence of Xoo on indica and japonica rice varieties. Our findings indicate that pathogen-induced sucrose efflux via SWEETs provides sucrose to Xoo, that Xoo uses the sux gene cluster to acquire and utilize sucrose, and that sucrose promotes bacterial fitness and xylem colonization.

Highlights: – Xoo produces filamentous morphology, which is transient and yields pleomorphic progenies in vitro – In planta , initial attachment of rod-shaped Xoo is detected at xylem pits – The Xoo infection front migrates basipetally in the vascular bundle and progresses laterally from major to minor veins via transverse veins – Xoo breaks out of the xylem vessels and enter the neighboring xylem parenchyma – Xoo assumes filamentous morphology that can traverse from the xylem across the bundle sheath into mesophyll tissue – Mobility in xylem vessels depends predominantly on rod-shaped Xoo, while infection of mesophyll tissue at later stages appears to be linked to filamentous morphology Summary: Xanthomonas oryzae pv. oryzae (Xoo) is classified as a xylem pathogen responsible for bacterial blight of rice causing substantial yield losses in Asia and Africa. Xoo virulence depends on the ability to trigger SWEET sucrose efflux transporters in the xylem parenchyma (XP) by injection of transcription activation like effectors (TALe) into host cells, likely to access host-derived sucrose. To establish infection, Xoo must overcome physical barriers, immune responses and the hydraulic xylem flow. To gain insights into the colonization process, we used translational SWEET11a-GUS reporter lines, scanning electron microscopy, and confocal laser scanning microscopy of Xoo tagged with a fluorescent protein. We found that Xoo can differentiate in vitro into filamentous forms. We mapped the infection route of Xoo along the vasculature, identified distinct spatiotemporal phases of Xoo colonization marked by rod-shaped and, notably, filamentous Xoo cells. Rod-shaped Xoo were found to attach to xylem pits during basipetal progression of the infection. Notably, we found that at later infection stages, Xoo could enter the XP. Strikingly, Xoo adopted a filamentous phenotype that traversed bundle sheath cells and entered mesophyll cells. Chlorosis and necrosis of leaves is thus likely not just due to blockage of xylem flow, but to direct tissue damage. Filamentation had been reported as important for virulence of human pathogens e.g. Yersinia pestis , uropathogenic E. coli and Shigella and had been associated to sugar utilization in Bacillus subtilis . We thus hypothesize that Xoo differentiation during host colonization is critical for virulence. Graphical

Controlled plant growth in laboratories can be achieved by cultivating plants in sterile or axenic conditions on pre-defined synthetic growth media typically supplemented with sugar. In nature, plants do not receive exogenous sugar supply, form symbiosis with microbes, and plant growth is influenced by soil edaphic factors. Thus, physiological and multi-omic analyses from plants grown on synthetic media will differ from those of soil-grown plants due to the influence of sucrose, and the lack of influence from microbiota and soil edaphic factors on plant growth. The rapid advancements in spatial-omics call for accurate characterization of plants grown in conditions similar to soil. To address the issue, we developed Artificial Soil (ArtSoil), a growth medium containing essential nutrients for plant growth, and aqueous soil extract (ASE) to maintain soil microbiomes and edaphic factors, simultaneously eliminating the need for sugar supplementation in the medium. We compared Arabidopsis thaliana grown on conventional media to ArtSoil under various growth conditions, and showed that complex soil microbiota in ArtSoil promote plant growth without physiological side effects induced by sucrose. We demonstrate an application for ArtSoil in single-cell transcriptomics and report microbiota-induced cell-type specificity in immune and nitrogen signaling. We tested ArtSoil with six types of ASEs to present the potential of ArtSoil in decoupling the effects of nutrients from microbiota in plant growth. We conclude that ArtSoil recapitulates the soil environment compared to conventional media, hence, enabling physiologically relevant plant growth.

Bacterial blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL‐effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) of SWEET promoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome‐edited (GE'd) rice mega‐varieties (IR64, Ciherang‐Sub1 for Asia and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GE'd lines, including evaluation of agronomic performance in multi‐location multi‐season experimental field plots under different fertilisation regimes and tests for the presence/absence of foreign DNA/transgene in the offspring of GE'd lines (IR64‐BC1T6, Ciherang‐Sub1‐BC1T5, Komboka‐T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country‐specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify the heritability of the edits, single‐nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE‐induced off‐target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang‐Sub1, and Komboka are provided. In the field experiments, the GE'd lines did not show performance defects. Together, the results indicate that select GE'd lines do not contain foreign DNA or transgene fragments and fulfil the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

Xanthomonas oryzae pv. oryzae ( Xoo ), the causal agent of bacterial blight in rice, is primarily studied in the context of foliar infections. However, infected stubble and irrigation water may serve as reservoirs and be responsible for seedling stage root infections in the field, especially during transplanting. Here, we established a coleoptile crown root-based infection protocol to investigate whether gene-for-gene interactions between Xoo TAL effectors and SWEET sucrose uniporter susceptibility genes occur in the root xylem, and whether the disease can propagate from roots to seedling shoots. Using translational SWEET11a-GUS reporter lines under control of the native SWEET11a promoter, we observed progressive infection as indicated by accumulation of SWEET11a-GUS fusion protein in infected coleoptile crown roots. However, we did not detect progression of GUS accumulation beyond the coleoptile node, nor did we detect blight symptoms on the young leaves. Notably, the xylem, at least during early stages of infection remained functional as shown by Rhodamine B tracer, consistent with transfer of xylem constituents via living cells at the coleoptile node that did not allow bacteria to pass. Furthermore, the root infection protocol is a ∼4x faster compared to standard leaf-clipping assays (roots assay: 11 days from sowing, compared to 39 days for clip infection), enabling more rapid assessment of TAL effector repertoire and plant defense responses with translational SWEET-GUS reporter lines. Our findings expand our understanding of Xoo infection routes and provide a valuable tool for resistance testing and pathogen surveillance.

A path to sustainably reduce world hunger, food insecurity, and malnutrition is to close the crop yield gap and, particularly, lower losses due to pathogens. Breeding resistant crops is key to achieving this goal, which is an effort requiring collaboration among stakeholders, scientists, breeders, farmers, and policymakers. During a disease outbreak, epidemiologists survey the occurrence of a disease after which pathologists investigate mechanisms to stop an infection. Policymakers then implement strategies with farmers and breeders to overcome the outbreak. Information flow from the field to the lab and back to the field involves several processing hubs that require different information inputs. Failure to communicate the necessary information results in the transfer of meaningless data. Here, we discuss gaps in information acquisition and transfer between the field and laboratory. Using rice bacterial blight disease as an example, we discuss pathogen biology and disease resistance to point out the importance of reporting pathogen strains that caused an outbreak to optimize the deployment of resistant crop varieties. We examine differences between infection in the field and assays performed in the laboratory to draw awareness of possible misinformation concerning plant resistance or susceptibility. We discuss key data considered useful for reporting disease outbreaks, sampling bias, and suggestions for improving data quality. We also touch on the knowledge gap in the state-of-the-art literature regarding disease dispersal and transmission. We use a recent case study to exemplify the gaps mentioned. We conclude by highlighting potential actions that may contribute to food security and to closing the yield gap. [ ] Copyright © 2025 The Author(s). This is an open access article distributed under the CC BY 4.0 International license .