Molecular Plant Pathology

ABSTRACT Ubiquitin‐proteasome is a conserved mechanism that regulates cellular responses and disease resistance in plants. However, the regulatory role of ubiquitin‐proteasome in the pathogenicity of “ Candidatus Liberibacter asiaticus” ( C Las), the causal agent of citrus huanglongbing (HLB), one of the most serious citrus diseases, remains poorly defined. In this study, we demonstrated that CLIBASIA_04250 (SDE4250) suppresses 26S proteasome activity in citrus, leading to a reduction in salicylic acid (SA) levels and downregulation of SA‐responsive genes. Further investigation revealed that SDE4250 specifically targets the citrus 26S proteasome non‐ATPase regulatory subunit 4 homologue (CsRPN10) and enhances its degradation via the 26S proteasome pathway. Moreover, overexpression of CsRPN10 inhibited C Las proliferation in citrus hairy roots, which was accompanied by a significant accumulation of SA content and upregulation of related defence genes. Collectively, our findings indicate that SDE4250 targets the citrus 26S proteasome to disrupt SA‐mediated citrus immunity.

ABSTRACT Citrus huanglongbing (HLB), caused by the phloem‐limited bacterium ‘ Candidatus Liberibacter asiaticus’ ( C Las), is one of the most destructive diseases in global citrus production. Here, we report the functional characterization of a core C Las effector, CLIBASIA_00525 named SDE525, that manipulates host immunity by targeting the citrus nascent polypeptide‐associated complex alpha subunit (CsNACα). We confirmed that SDE525 is a secreted protein that localizes to the nucleus, cytoplasm, and plasma membrane. Notably, the effect of SDE525 exhibits host‐specific context dependence: while its transient expression in the non‐host Nicotiana benthamiana unexpectedly triggered immune responses, in its native host, sweet orange ( Citrus sinensis ), SDE525 interacted with CsNACα to modulate defence, as demonstrated by multiple independent assays. Integrated transcriptomic and metabolomic analyses revealed that this interaction reprograms the host's hormonal landscape, suppressing the jasmonic acid (JA) signalling pathway while simultaneously activating the salicylic acid (SA) pathway. Importantly, under transient overexpression conditions in citrus, this immune reprogramming enhanced resistance against the bacterial canker pathogen Xanthomonas citri subsp. citri —a phenomenon distinct from the susceptibility observed during authentic C Las infection. Our findings uncover a sophisticated mechanism by which a C Las effector rewires host defence signalling, providing fundamental insights into its pathogenesis with potential long‐term implications for future HLB management.

ABSTRACT Citrus huanglongbing (HLB), caused by “ Candidatus Liberibacter” spp., is one of the most disastrous citrus diseases worldwide. HLB‐affected citrus fruits are significantly more acidic than healthy fruits. However, the molecular mechanism behind this phenomenon remains to be elucidated. Here, we report that HLB‐affected fruits have higher levels of citric acid (CA) than healthy fruits. Moreover, Citrus PH4 ( CitPH4 ), which encodes a MYB transcription factor that functions as a key regulator of CA accumulation, was upregulated in HLB‐affected fruits relative to healthy fruits. Heterologous overexpression of CitPH4 in tobacco ( Nicotiana tabacum ) plants enhanced tolerance to HLB. Subsequently, overexpression and gene‐editing experiments indicated that CitPH4 can affect the salicylic acid (SA) pathway, which directly binds to and activates the promoter of CsPBS3 , a key gene of SA biosynthesis. HLB‐affected fruits had higher SA levels than healthy fruits. Furthermore, application of SA activated CA biosynthesis and application of CA activated SA biosynthesis and signalling in citrus fruits and decreased “ Candidatus Liberibacter asiaticus” ( C Las) titres in infected leaves. This work suggests that CitPH4 is a key node between CA and SA, thus revealing crosstalk between defence responses and fruit quality in citrus.

Abstract Citrus huanglongbing (HLB) has been causing enormous damage to the global citrus industry. As the main causal agent, ‘ Candidatus Liberibacter asiaticus’ ( C Las) delivers a set of effectors to modulate host responses, while the modes of action adopted remain largely unclear. Here, we demonstrated that CLIBASIA_00185 ( C Las0185) could attenuate reactive oxygen species (ROS)‐mediated cell death in Nicotiana benthamiana . Transgenic expression of C Las0185 in Citrus sinensis ‘Wanjincheng’ enhanced plant susceptibility to C Las. We found that methionine sulphoxide reductase B1 (CsMsrB1) was targeted by the effector, and its abundance was elevated in CLas0185 ‐transgenic citrus plants. Their interaction promoted C Las proliferation. We then determined that CsMsrB1 sustained redox state and enzymatic activity of ascorbate peroxidase 1 (CsAPX1) under oxidative stress. The latter reduced H 2 O 2 accumulation and was associated with host susceptibility to C Las infection. Consistently, citrus plants expressing C Las0185 and CsMsrB1 conferred enhanced APX activity and decreased H 2 O 2 content. Taken together, these findings revealed how C Las0185 benefits C Las colonization by targeting CsMsrB1, which facilitated the antioxidant activity and depressed ROS during pathogen infection.

Abstract Citrus huanglongbing (HLB), associated with the unculturable phloem‐limited bacterium “ Candidatus Liberibacter asiaticus” ( C Las), is the most devastating disease in the citrus industry worldwide. However, the pathogenicity of C Las remains poorly understood. In this study, we show that AGH17488, a secreted protein encoded by the prophage region of the C Las genome, suppresses plant immunity via targeting the host ASCORBATE PEROXIDASE6 (APX6) protein in Nicotiana benthamiana and Citrus sinensis . The transient expression of AGH17488 reduced the chloroplast localization of APX6 and its enzyme activity, inhibited the accumulation of reactive oxygen species (H 2 O 2 and O 2 − ) and the lipid oxidation endproduct malondialdehyde in plants, and promoted the proliferation of Pseudomonas syringae pv. tomato DC3000 and Xanthomonas citri subsp. citri . This study reveals a novel mechanism underlying how C Las uses a prophage‐encoded effector, AGH17488, to target a reactive oxygen species accumulation‐related gene, APX6 , in the host to facilitate its infection.

Abstract Huanglongbing (HLB), associated with “ Candidatus Liberibacter asiaticus” (CLas), is a globally devastating plant disease. The highly reduced genome of CLas encodes a number of secretory proteins. The conserved prophage‐encoded protein AGH17470 is herein identified as a nonclassical secretory protein. We confirmed that the N‐terminal and C‐terminal sequences jointly determine the secretion of AGH17470. The transient expression of AGH17470 protein in Nicotiana benthamiana caused hypersensitive response (HR) cell death in infiltrated leaves and systemically infected leaves as well as the dwarfing of the entire plant, suggesting that AGH17470 is involved in the plant immune response, growth, and development. Overexpression of AGH17470 in N. benthamiana and citrus plants up‐regulated the transcription of pathogenesis‐related and salicylic acid (SA)‐signalling pathway genes and promoted SA accumulation. Furthermore, transient expression of AGH17470 enhanced the resistance of sweet orange to Xanthomonas citri subsp. citri . To our knowledge, AGH17470 is the first prophage‐encoded secretory protein demonstrated to elicit an HR and induce a strong plant immune response. The findings have increased our understanding of prophage‐encoded secretory protein genes, and the results provide clues as to the plant defence response against CLas.

Summary Citrus huanglongbing (HLB) is the most devastating citrus disease worldwide. ‘ Candidatus Liberibacter asiaticus’ (Las) is the most prevalent HLB causal agent that is yet to be cultured. Here, we analysed the flagellar genes of Las and Rhizobiaceae and observed two characteristics unique to the flagellar proteins of Las: (i) a shorter primary structure of the rod capping protein FlgJ than other Rhizobiaceae bacteria and (ii) Las contains only one flagellin‐encoding gene flaA (CLIBASIA_02090), whereas other Rhizobiaceae species carry at least three flagellin‐encoding genes. Only flgJ Atu but not flgJ Las restored the swimming motility of Agrobacterium tumefaciens flgJ mutant. Pull‐down assays demonstrated that FlgJ Las interacts with FlgB but not with FliE. Ectopic expression of flaA Las in A. tumefaciens mutants restored the swimming motility of ∆ flaA mutant and ∆ flaAD mutant, but not that of the null mutant ∆ flaABCD . No flagellum was observed for Las in citrus and dodder. The expression of flagellar genes was higher in psyllids than in planta . In addition, western blotting using flagellin‐specific antibody indicates that Las expresses flagellin protein in psyllids, but not in planta . The flagellar features of Las in planta suggest that Las movement in the phloem is not mediated by flagella. We also characterized the movement of Las after psyllid transmission into young flush. Our data support a model that Las remains inside young flush after psyllid transmission and before the flush matures. The delayed movement of Las out of young flush after psyllid transmission provides opportunities for targeted treatment of young flush for HLB control.

SUMMARY Phytoplasmas are plant‐pathogenic bacteria that are associated with numerous plant diseases. We have previously reported the complete genomic sequence of Candidatus Phytoplasma asteris, OY strain, OY‐M line, which causes mild symptoms. The phytoplasma genome lacks several important metabolic genes, implying that the consumption of metabolites by phytoplasmas in plants may cause disease symptoms. Here we show that the approximately 30‐kb region including the glycolytic genes was tandemly duplicated in the genome of OY‐W phytoplasma, which causes severe symptoms. Almost duplicated genes became pseudogenes by frameshift and stop‐codon mutations, probably because of their functional redundancy. However, five kinds of genes, including two glycolytic genes, remained full‐length ORFs, suggesting that it is advantageous for the phytoplasma to retain these genes in its lifestyle. In particular, 6‐phosphofructokinase is known as a rate‐limiting enzyme of glycolysis, implying that the different number of glycolytic genes between OY‐W and OY‐M may influence their respective glycolysis activities. We previously reported that the phytoplasma population of OY‐W was higher than that of OY‐M in their infected plants. Taking this result into account, the higher consumption of the carbon source may affect the growth rate of phytoplasmas and also may directly or indirectly cause more severe symptoms.