Pathogens from the fastidious, phloem-restricted 'Candidatus Liberibacter' species cause the devastating Huanglongbing (HLB) disease in citrus worldwide and cause diseases on many solanaceous crops and plants in the Apiaceae family. However, little is known about the pathogenic mechanisms due to the difficulty in culturing the corresponding 'Ca. Liberibacter' species. Here, we report that the citrus HLB pathogen 'Ca. L. asiaticus' uses an active salicylate hydroxylase SahA to degrade salicylic acid (SA) and suppress plant defenses. Purified SahA protein displays strong enzymatic activity to degrade SA and its derivatives. Overexpression of SahA in transgenic tobacco plants abolishes SA accumulation and hypersensitive response (HR) induced by nonhost pathogen infection. By degrading SA, 'Ca. L. asiaticus' not only enhances the susceptibility of citrus plants to both nonpathogenic and pathogenic Xanthomonas citri but also attenuates the responses of citrus plants to exogenous SA. In addition, foliar spraying of 2,1,3-benzothiadiazole and 2,6-dichloroisonicotinic acid, SA functional analogs not degradable by SahA, displays comparable (and even better) effectiveness with SA in suppressing 'Ca. L. asiaticus' population growth and HLB disease progression in infected citrus trees under field conditions. This study demonstrates one or more pathogens suppress plant defenses by degrading SA and establish clues for developing novel SA derivatives-based management approaches to control the associated plant diseases.
Rice yellow stunt virus (RYSV) is a member of the plantinfecting Nucleorhabdovirus genus of the Rhabdoviridae family (35). RYSV has a nonsegmented, negative-sense single-stranded RNA genome which contains a 5Ј trailer, a 3Ј leader, and seven open reading frames (ORFs) in the order
Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. HLB is associated with the non-culturable bacterium, Candidatus Liberibacter asiaticus (CaLas) in the United States. The virulence mechanism of CaLas is largely unknown, partly because of the lack of a mutant library. In this study, Tobacco mosaic virus (TMV) and Nicotiana benthamiana (N. benthamiana) were used for large-scale screening of the virulence factors of CaLas. Agroinfiltration of 60 putative virulence factors in N. benthamiana led to the identification of four candidates that caused severe symptoms in N. benthamiana, such as growth inhibition and cell death. CLIBASIA_05150 and CLIBASIA_04065C (C-terminal of CLIBASIA_04065) could cause cell death in the infiltrated leaves at five days post infiltration. Two low-molecular-weight candidates, CLIBASIA_00470 and CLIBASIA_04025, could inhibit plant growth. By converting start codon to stop codon or frameshifting, the four genes lost their harmful effects to N. benthamiana. It indicated that the four virulence factors functioned at the protein level rather than at the RNA level. The subcellular localization of the four candidates was determined by confocal laser scanning microscope. CLIBASIA_05150 located in the Golgi apparatus; CLIBASIA_04065 located in the mitochondrion; CLIBASIA_00470 and CLIBASIA_04025 distributed in cells as free GFP. The host proteins interacting with the four virulence factors were identified by yeast two-hybrid. The host proteins interacting with CLIBASIA_00470 and CLIBASIA_04025 were overlapping. Based on the phenotypes, the subcellular localization and the host proteins identified by yeast two-hybrid, CLIBASIA_00470 and CLIBASIA_04025, functioned redundantly. The hypothesis of CaLas virulence was proposed. CaLas affects citrus development and suppresses citrus disease resistance, comprehensively, in a complicated manner. Ubiquitin-mediated protein degradation might play a vital role in CaLas virulence. Deep characterization of the interactions between the identified virulence factors and their prey will shed light on HLB. Eventually, it will help in developing HLB-resistant citrus and save the endangered citrus industry worldwide.
Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.
Summary
In plants, exogenous transgene transcribing inverted-repeat (exo-IR) sequences produces double-stranded RNAs that are processed by DCL4. The generated 21-nt siRNAs function as mobile signals to trigger non-cell-autonomous silencing of target endogene in neighboring 10-15 cells. The potential involvement of nuclear silencing pathway components in signal spreading or sensing in target cells is not clear. Here, we demonstrate that the exo-IR silencer (exo-Pdsi) is negatively auto-regulated through methylation spreading, which acts in cis to reinforce self-silencing of the silencer. Mutations affecting nuclear proteins DRD1 and Pol V (NRPE1 or NRPD2) relieved exo-Pdsi self-silencing resulting in higher levels of Pdsi transcripts which increased non-cell-autonomous silencing of endo-PDS. Our results suggest that in an experimental silencing pathway, methylation spreading on a silencer transgene may not have a direct endogenous plant counterpart when protein-encoding gene is the target. DRD1-Pol V-dependent de novo methylation, by acting in cis to reinforce self-silencing of exo-IR may play a role in restraining inappropriate silencing of active protein-coding genes in plants.
Huanglongbing (HLB), also known as citrus greening, is the most destructive disease of citrus worldwide. In the United States, this disease is associated with a phloem-restricted bacterium, Candidatus Liberibacter asiaticus. Commercial citrus cultivars are susceptible to HLB, but Poncirus trifoliata, a close relative of Citrus, is highly tolerant of HLB. Isolating P. trifoliata gene(s) controlling its HLB tolerance followed by expressing the gene(s) in citrus is considered a potential cisgenic approach to engineering citrus for tolerance to HLB. Previous gene expression studies indicated that the constitutive disease resistance (CDR) genes in P. trifoliata (PtCDRs) may play a vital role in its HLB tolerance. This study was designed to use Arabidopsis mutants as a model system to confirm the function of PtCDRs in plant disease resistance. PtCDR2 and PtCDR8 were amplified from P. trifoliata cDNA and transferred into the Arabidopsis cdr1 mutant, whose resident CDR1 gene was disrupted by T-DNA insertion. The PtCDR2 and PtCDR8 transgenic Arabidopsis cdr1 mutant restored its hypersensitive response to the bacterial pathogen Pseudomonas syringae pv. tomato strain DC3000 (Pst DC3000) expressing avrRpt2. The defense marker gene PATHOGENESIS RELATED 1 (PR1) expressed at much higher levels in the PtCDR2 or PtCDR8 transgenic cdr1 mutant than in the non-transgenic cdr1 mutant with or without pathogen infection. Multiplication of Pst DC3000 bacteria in Arabidopsis was inhibited by the expression of PtCDR2 and PtCDR8. Our results showed that PtCDR2 and PtCDR8 were functional in Arabidopsis and played a positive role in disease resistance and demonstrated that Arabidopsis mutants can be a useful alternate system for screening Poncirus genes before making the time-consuming effort to transfer them into citrus, a perennial woody plant that is highly recalcitrant for Agrobacterium or biolistic-mediated transformation.
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