Publications

4869

SUMMARY The ubiquitin proteasome system (UPS) and autophagy cooperatively orchestrate plant immune homeostasis, while diverse phytopathogens deploy secreted effectors to disrupt their crosstalk for successful colonization. Proteaphagy, the autophagic turnover of intact 26S proteasome holoenzymes, is a conserved stress response co‐opted by Pseudomonas syringae effector HopM1 to cripple host proteolysis. However, how the Huanglongbing (HLB) pathogen ‘ Candidatus Liberibacter asiaticus’ ( C Las) simultaneously manipulates UPS and autophagy to subvert citrus immunity remains largely uncharacterized. Here, we functionally characterize the secreted C Las effector SDE5640, which exacerbates HLB susceptibility in sweet orange ( Citrus sinensis ). SDE5640 directly binds the 19S proteasome chaperone CsPSMD10, whose homodimerization via Cys163/Cys190 disulfide bonds is required for immune suppression. CsPSMD10 overexpression enhances citrus susceptibility to C Las, whereas the dimerization‐deficient CsPSMD10‐C190S mutant confers robust HLB resistance. Mechanistically, CsPSMD10 targets CsATG8 isoforms for UPS‐dependent degradation to restrain autophagic flux; SDE5640 competitively occupies the Ank2/Ank3 domains of CsPSMD10 to disrupt CsPSMD10‐CsATG8c complexes, thereby stabilizing CsATG8c and triggering autophagy dependent on CsPSMD10. Moreover, SDE5640‐induced autophagy drives selective autophagic degradation of the 20S proteasome catalytic subunit CsPBA1, which ultimately disrupts cellular proteasome activity and attenuates salicylic acid‐mediated citrus immunity. Collectively, our findings identify a novel SDE5640‐CsPSMD10‐CsATG8c‐CsPBA1 regulatory cascade, illustrating how a single C Las effector synchronously rewires the UPS‐autophagy crosstalk to facilitate pathogen proliferation.

Abstract Antimicrobial peptides (AMPs) are widely used in disease-resistant breeding, and their application in citrus has recently been reported. However, the molecular mechanism by which AMPs inhibit the Huanglongbing (HLB) remains unclear. This study aimed to clarify the inhibitory mechanism of Hinnavin I (an AMP) against HLB-associated pathogens. We first demonstrated that Hinnavin I exhibits antibacterial activity against several tested Gram-negative bacterial strains. Using a transgenic citrus hairy root system, the heterologous expression of Hinnavin I was found to significantly reduce Candidatus Liberibacter asiaticus (CLas) colonization. Transcriptomic and phytohormone analyses further showed that constitutive Hinnavin I expression was associated with altered defense-related gene expression and enhanced SA-related responses in citrus hairy roots. Multiple interaction assays identified the CLas outer membrane lipoprotein Omlp as a direct interaction partner of Hinnavin I both in vitro and in vivo.

Abstract Purpose The objective of this study was to characterize Bradyrhizobium strains currently recommended as inoculants for Arachis pintoi in Brazil using comparative genomic and symbiotic analyses. In addition, this study aimed to expand current knowledge on the diversity, evolution, and bacterial symbiotic traits of Arachis -associated Bradyrhizobium while providing robust taxonomic evidence to support the description of a novel Bradyrhizobium species isolated from root nodules of Arachis . Methods Phylogenetic reconstruction was performed based on the 16S rRNA gene and by phylogenomics using 116 single- copy core gene sequences. Average Nucleotide Identity (ANI) analyses were conducted to evaluate the genomic relatedness of strains BR 1432ᵀ, BR 1433 and BR 14999 to 1,301 reference Bradyrhizobium genomes, including all available type strain strains. The results were validated using digital DNA–DNA hybridization (dDDH). Additional analyses included phylogenetic reconstruction of symbiotic genes ( nodC and nifH ) and, assessment of the denitrification pathway, and phenotypic characterization. Results Phylogenetic analyses based on 16S rRNA gene sequences and whole-genome data indicated that strains BR 1432ᵀ and BR 14999 form a distinct lineage within the genus Bradyrhizobium , clearly separated from BR 1433, which clustered with Bradyrhizobium brasilense . ANI and dDDH values between BR 1432ᵀ and BR 14999 and their closest related type strains, including B. zhanjiangense LMG 29279ᵀ, were below the species delineation thresholds of 96% and 70%, respectively, supporting the proposal of a novel species. In addition, the publicly available RefSeq genomes Bradyrhizobium sp. DN5 and Rc2d were found to be closely related to BR 1432ᵀ. Phylogenetic analyses of the nodC and nifH genes revealed distinct evolutionary patterns, while genomic analyses showed that the proposed novel species harbors a complete denitrification pathway, unlike strain BR 1433. Furthermore, phenotypic characterization revealed differences in carbon source utilization and enzymatic activities. Conclusion Based on genomic, phylogenetic, and phenotypic evidence, strain BR 1433 was identified as a member of B. brasilense , whereas strains BR 1432ᵀ (= UCCC 277ᵀ = LMG 34120ᵀ = CCP577ᵀ), BR 14999, DN5 and Rc2d represent a novel species within the genus Bradyrhizobium , for which the name Bradyrhizobium boddeyi sp. nov. is proposed.

Huanglongbing (aka: citrus greening) associated with ‘Candidatus Liberibacter asiaticus’ (CLas) is one of the most impactful citrus diseases throughout the world. The aim of this project was to support the sensitive and rapid detection of CLas to prevent the continued spread and destruction of US citrus crops by this psyllid vector-borne disease. Two multicopy targets specific to CLas were selected for assay development: the five-copy nrdB (ribonucleotide reductase-RNR) gene and a four-copy locus 4cp (4CP). Published RNR RPA primers were used to develop a one-pot RPA-CRISPR assay, and RPA primers for 4CP were designed. crRNAs for both RNR- and 4CP-RPA-CRISPR/Cas assays were designed, evaluated through preliminary testing, and the best candidate primers and crRNA combinations were selected. The 4CP assay was selected and subjected to a battery of validation tests and determined fit-for-purpose. A field extraction protocol was also developed using commercial reagents, syringes and filters to bring kit sensitivity to the field. When combined with the assay, the complete workflow was able to detect CLas in infected citrus, down to a single infected leaf per subsample; the field workflow showed similar sensitivity to the laboratory-based workflow. While the extraction and assay proved useful, particularly for symptomatic leaves, its performance with asymptomatic leaves for early detection was not evaluated. The complexity of the assay in its current form hinders widespread practical applicability. In the future, revalidation of lyophilized reagents in a convenient kit can be performed for practical deployment; preliminary testing has shown success.