Biology subjects

Schepler-Luu, V.

Publications and source records attributed to Schepler-Luu, V..

6 recordsLinked to original sources

bioRxiv2026

Natural variation in the atypical resistance gene Pita2 confers broad-spectrum neck blast resistance in rice

Neck blast (NB), caused by Magnaporthe oryzae, damages rice panicles and reduces yield. Knowledge of NB resistance remains limited due to the lack of reliable resistance evaluation methods. Here, we applied a newly established neck injection method and performed a GWAS on 335 diverse accessions from the 3K Rice Genomes Project to identify loci associated with NB resistance. We detected a significant association on chromosome 12, explaining 15-18% of the symptom variations caused by a highly virulent Philippine blast isolate (M64-1-3-9-1). Linkage disequilibrium analysis refined this region to a 42.3-kb interval containing Pita2, a known leaf blast resistance gene. We found that two Pita2 allelic variants, Pita2a and Pita2c, both harboring the variant A/G (Lys879) in the last exon (Chr12:10,833,400), are associated with NB resistance. IR64 and a CO39 near-isogenic line (NIL) IRBLta2-Pi[CO] harboring Pita2a were resistant, whereas CRISPR-Cas9 knock-out of Pita2a in IR64 caused susceptibility to M64-1-3-9-1 and IK81-25. These results indicate that Pita2a is required for NB resistance. Furthermore, the CO39 NIL, IRBLta2-Pi[CO], and Lijiangxintuanheigu monogenic line (IRBLta2-Pi) harboring the Pita2a allele exhibited broad-spectrum resistance to 75% and 80% of Philippine differential blast isolates, respectively. The superior haplotype of Pita2 contains two major SNPs (A/G and A/C at Chr12:10,833,400 and Chr12:10,845,095) occurs in 83% of IRRI elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings identify Pita2a as a major gene for NB resistance and provide a valuable genetic resource for developing blast-resistant rice. PLAIN LANGUAGE SUMMARYRice blast, caused by the fungus Magnaporthe oryzae, is a major threat to global rice production. Neck blast (NB) is the most severe type of blast, however, the genetic basis of NB resistance remains poorly understood. In this study, we analyzed 335 rice accessions to identify genes underlying the resistance against a Philippine blast isolate. We found that allelic variants Pita2a and Pita2c are strongly-associated with NB resistance. Knock-out of Pita2a allele made resistant rice plants susceptible while introgression into susceptible rice lines enhanced resistance to multiple blast isolates, confirming its role in NB resistance. Importantly, the superior alleles of Pita2 are already present in 83% of elite breeding lines and can be used to select NB-resistant genotypes with an accuracy of 86%. Our findings clarify the genetic control of NB resistance and offer new tools for protecting rice yields in blast-endemic regions.

pathology↗

bioRxiv2025

Differentiation of Xanthomonas oryzae pv. oryzae in vitro and during rice leaf infection

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 SummaryXanthomonas 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 abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/680524v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@f66d8borg.highwire.dtl.DTLVardef@91ca66org.highwire.dtl.DTLVardef@17f2846org.highwire.dtl.DTLVardef@12d46e7_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

From lab to field: analyses of genome-edited bacterial blight resistant rice

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 (GEd) 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 GEd lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GEd 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 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 GEd lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

plant biology↗

bioRxiv2023

Loss-of-function mutation in the polyamine transporter gene OsLAT5 as a selectable marker for genome editing

Genome editing by TALENs, CRISPR/Cas, base or prime editing have become routine tools. During stable plant transformation, the gene coding for the editing enzyme, e.g., Cas9, the guide RNAs (gRNAs), alongside a selectable marker are integrated into the nuclear genome. Identification of successful transformants relies on selectable or screenable markers, typically genes providing resistance to antibiotics or herbicides. Selectable markers use a substantial portion of the T-DNA, hence reducing transfer efficiency by limiting the effective number of TALENs or guide/pegRNAs that can be used in parallel. Moreover, marker genes are frequently subject to gene silencing. Here, we generated loss-of-function mutations in PUT/LAT-type polyamine transporter family genes to confer resistance to the phytotoxin methylviologen (MV) as a method for selection. As a proof of concept, CRISPR/Cas9 vectors with gRNAs were constructed to target three close homologs OsLAT1, OsLAT5, and OsLAT7. We show that loss of OsLAT5 (also known as OsPUT3 or OsPAR1) function was sufficient to confer resistance to MV in rice seeds, seedlings and calli, validating the editing approach of OsLAT5 to obtain a selectable marker. We discuss the potential of incorporating a gRNA cassette (for OsLAT5) as a selectable marker and a reporter for successful genome editing for optimizing editing protocols.

plant biology↗

bioRxiv2023

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (O. glaberrima), however, introgression into O. sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role in RYMV susceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single knockout mutants show neither substantial growth defects nor symptoms indicative of programmed cell death, possibly reflecting functional redundancy of the isoforms regarding other important functions. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

plant biology↗

bioRxiv2022

Genome editing of an African elite rice variety confers resistance against endemic and emerging Xanthomonas oryzae pv. oryzae strains

Bacterial leaf blight (BB) of rice, caused by Xanthomonas oryzae pv. oryzae (Xoo), threatens global food security and the livelihood of small-scale rice producers. Analyses of Xoo collections from Asia, Africa and the Americas demonstrated surprising continental segregation, despite robust global rice trade. Here, we report unprecedented BB outbreaks in Tanzania. The causative strains, unlike endemic Xoo, carry Asian-type TAL effectors targeting the sucrose transporter SWEET11a and suppressing Xa1. Phylogenomics clustered these strains with Xoo strains from China. African rice varieties do not carry suitable resistance genes. To protect African rice production against this emerging threat, we developed a hybrid CRISPR-Cas9/Cpf1 system to edit six TALe-binding elements in three SWEET promoters of the East African elite variety Komboka. The edited lines show broad-spectrum resistance against Asian and African strains of Xoo, including strains recently discovered in Tanzania. This strategy could help to protect global rice crops from BB pandemics.

plant biology↗