Laixin Luo

Bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most serious bacterial disease that seriously reduces the yield of rice. Previous studies showed that insertion of effector binding elements (EBEs) into the Xa23 promoter can confer resistance to various Xanthomonas oryzae pv. oryzae in O. sativa japonica, a model cultivar for research purposes. Therefore, it is necessary to edit elite varieties to test whether this strategy works in rice production. In this research, the grand prime gene editing (PE) method was used to efficiently knock in four consecutive EBE sequences in the promoter region of the resistance gene Xa23 of the elite variety Suijing18. A PE vector was constructed and transformed into rice calli, and transgene-free homozygously edited plants were identified. Bioassay results show that the edited plants exhibited a significantly higher level of resistance to two strains of Xoo, PXO86 and PXO99A, than the wild-type control. This study demonstrated that Grand PE could efficiently introduce resistance to bacterial blight in elite varieties.

Impact statement: Antibiotic resistance in plant pathogens threatens sustainable crop protection, and yet, its molecular basis remains incompletely understood. We report that streptomycin resistance in Clavibacter michiganensis strain TX‐0702 is mediated by a plasmid‐borne aminoglycoside phosphotransferase, APH(3), encoded on an uncharacterized plasmid, pCM3. Functional and biochemical analyses demonstrate that APH(3) inactivates streptomycin through phosphorylation, establishing a phosphorylation‐driven resistance mechanism in Gram‐positive plant pathogens. Sequence analyses reveal that pCM3 carries mobile genetic elements, suggesting environmental dissemination of resistance genes in plant‐associated microbes. These findings expand understanding of phytopathogen antibiotic resistance and plant microbiomes as potential resistance gene sources for agricultural, food, and human health safety.

The gram-positive bacterium Clavibacter michiganensis (Cm) causes bacterial canker of tomato. The infection process of Cm is of considerable interest, and the role of the alarmone guanosine tetraphosphate or pentaphosphate [(p)ppGpp], a global regulator, has been strongly implicated in bacterial survival and pathogenicity. Transcriptome analysis comparing a (p)ppGpp-deficient strain (Δ rel ) to the wild-type strain demonstrated that (p)ppGpp down-regulates the expression of many genes encoding ribosomal components and ABC transporter proteins, while up-regulating genes associated with amino acid metabolism, biofilm synthesis, and the production and secretion of cell-wall degrading enzymes. Biochemical assays showed reduced biofilm synthesis and extracellular activity of cell-wall degrading enzymes such as amylase and xylanase. Moreover, the results of microscale thermophoresis and electrophoretic mobility shift assays indicated that (p)ppGpp interacts with the transcription factor Vatr1 to directly suppress expression of the xylanase gene xysB . Meanwhile, amidase reporter assays revealed that xylanase secretion depends on the Tat secretion system, which was significantly inhibited in Δ rel , leading to intracellular accumulation of the xylanase protein. Taken together, these results indicate that (p)ppGpp plays a complex role in the pathogenicity of C. michiganensis , mediating, among other things, not only the production of cell-wall degrading enzymes but also their transport and secretion. IMPORTANCE: This study reveals the pivotal role of the bacterial alarmone guanosine tetraphosphate and guanosine pentaphosphate in the pathogenicity of Clavibacter michiganensis , a significant plant pathogen. Through the identification of its dual mechanisms in regulating enzyme production and the Tat secretion system, we uncover key insights into bacterial virulence strategies. Our findings not only advance the understanding of bacterial stress response systems but also offer new opportunities for developing targeted interventions to combat plant bacterial diseases, ultimately contributing to agricultural sustainability and food security.

Fungal phytopathogens employ effector proteins and secondary metabolites to subvert host immunity. Effector proteins have attracted widespread interest in infection, especially for unknown, unreported genes. However, the type of protein remains much less explored. Here, we combined transcriptome analysis and functional validation to identify virulence-associated genes in Fusarium graminearum during fungi infection. A unique secreted protein, FGSE02, was significantly upregulated in the early infection stage. Proteomic characterization revealed that the protein contains a functional signal peptide but lacks known domains. The transient expression of FGSE02 in Nicotiana benthamiana induced rapid cell death, while gene knockout stains reduced fungal virulence without affecting growth. Our findings highlight FGSE02 as a key virulence factor, offering potential targets for disease control. Taken together, the results of this study identify a pathogenic factor and provide new insights into the development of green pesticides.

Bacterial leaf spot of pepper (BSP), primarily caused by Xanthomonas euvesicatoria (Xe ), poses a significant challenge to pepper production worldwide. Despite its impact, the genetic diversity of this pathogen remains underexplored, which limits our understanding of its population structure. To bridge this knowledge gap, we conducted a comprehensive analysis using 103 Xe strains isolated from pepper in southwest Florida to characterize genomic and type III effector (T3E) variation in this population. Phylogenetic analysis of core genomes revealed a major distinct genetic lineage associated with amylolytic activity. This amylolytic lineage was represented in Xe strains globally. Molecular clock analysis dated the emergence of amylolytic strains in Xe to around 1972. Notably, non-amylolytic strains possessed a single base pair frameshift deletion in the ⍺-amylase gene yet retained a conserved C-terminus. GUS assay revealed the expression of two open reading frames in non-amylolytic strains, one at the N-terminus and another that starts 136 base pairs upstream of the ⍺-amylase gene. Analysis of T3Es in the Florida Xe population identified variation in 12 effectors, including two classes of mutations in avrBs2 that prevent AvrBs2 from triggering a hypersensitive response in Bs2 -resistant pepper plants. Knowledge of T3E variation could be used for effector-targeted disease management. This study revealed previously undescribed population structure in this economically important pathogen. IMPORTANCE: Bacterial leaf spot (BSP), a significant threat to pepper production globally, is primarily caused by Xanthomonas euvesicatoria ( Xe ). Limited genomic data has hindered detailed studies on its population diversity. This study analyzed the whole-genome sequences of 103 Xe strains from peppers in southwest Florida, along with additional global strains, to explore the pathogen’s diversity. The study revealed two major distinct genetic groups based on their amylolytic activity, the ability to break down starch, with non-amylolytic strains having a mutation in the ⍺-amylase gene. Additionally, two classes of mutations in the avrBs2 gene were found, leading to susceptibility in pepper plants with the Bs2 resistance gene, a commercially available resistance gene for BSP. These findings highlight the need to forecast the emergence of such strains, identify genetic factors for innovative disease management, and understand how this pathogen evolves and spreads.

Melia azedarach is a species of enormous value of pharmaceutical industries. Although the chloroplast genome of M. azedarach has been explored, the information of mitochondrial genome (Mt genome) remains surprisingly limited. In this study, we used a hybrid assembly strategy of BGI short-reads and Nanopore long-reads to assemble the Mt genome of M. azedarach. The Mt genome of M. azedarach is characterized by two circular chromosomes with 350,142 bp and 290,387 bp in length, respectively, which encodes 35 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes. A pair of direct repeats (R1 and R2) were associated with genome recombination, resulting in two conformations based on the Sanger sequencing and Oxford Nanopore sequencing. Comparative analysis identified 19 homologous fragments between Mt and chloroplast genome, with the longest fragment of 12,142 bp. The phylogenetic analysis based on PCGs were consist with the latest classification of the Angiosperm Phylogeny Group. Notably, a total of 356 potential RNA editing sites were predicted based on 35 PCGs, and the editing events lead to the formation of the stop codon in the rps10 gene and the start codons in the nad4L and atp9 genes, which were verified by PCR amplification and Sanger sequencing. Taken together, the exploration of M. azedarach gap-free Mt genome provides a new insight into the evolution research and complex mitogenome architecture.

Water hyacinth (Pontederia crassipes Mart.) is a monocotyledonous aquatic plant renowned for its rapid growth, extensive proliferation, biological invasiveness, and ecological resilience to variations in pH, nutrients, and temperature. The International Union for Conservation of Nature (IUCN) has listed P. crassipes among the top 100 invasive species. However, comprehensive genomic information, particularly concerning its mitochondrial genome (mitogenome), remains surprisingly limited. In this study, the complete mitogenome of P. crassipes was analyzed using bioinformatics approaches. The mitogenome is 399,263 bp long and contains 38 protein-coding genes (PCGs), 24 tRNA genes, and 3 rRNA genes. Sequence analysis revealed that the complete mitogenome of the species contains 3,289 dispersed repeats, and 765 RNA editing sites in protein-coding genes. The P. crassipes mitogenome possessed un-conserved structures, including extensive sequence transfer between its chloroplasts and mitochondria. Our study on the mitogenome of P. crassipes offers critical insights into its evolutionary patterns and phylogenetic relationships with related taxa. This research enhances our understanding of this invasive species, known for its significant biomass and rapid overgrowth in aquatic environments.