Publications

4857

ABSTRACT “ Candidatus Liberibacter asiaticus” (CLas), the causal agent of citrus Huanglongbing (HLB), is able to multiply to a high abundance in citrus fruit pith. However, little is known about the biological processes and phytochemical substances that are vital for CLas colonization and growth in fruit pith. In this study, CLas-infected fruit pith of three citrus cultivars (“Shatangju” mandarin, “Guanxi” pomelo, and “Shatian” pomelo) exhibiting different tolerance to CLas were collected and used for dual RNA-Seq and untargeted metabolome analysis. Comparative transcriptome analysis found that the activation of the CLas noncyclic TCA pathway and pathogenic-related effectors could contribute to the colonization and growth of CLas in fruit pith. The pre-established Type 2 prophage in the CLas genome and the induction of its CRISPR/ cas system could enhance the phage resistance of CLas and, in turn, facilitate CLas population growth in fruit pith. CLas infection caused the accumulation of amino acids that were correlated with tolerance to CLas. The accumulation of most sugars and organic acids in CLas-infected fruit pith, which could be due to the phloem blockage caused by CLas infection, was thought to be beneficial for CLas growth in localized phloem tissue. The higher levels of flavonoids and terpenoids in the fruit pith of CLas-tolerant cultivars, particularly those known for their antimicrobial properties, could hinder the growth of CLas. This study advances our understanding of CLas multiplication in fruit pith and offers novel insight into metabolites that could be responsible for tolerance to CLas or essential to CLas population growth. IMPORTANCE Citrus Huanglongbing (HLB, also called citrus greening disease) is a highly destructive disease currently threatening citrus production worldwide. HLB is caused by an unculturable bacterial pathogen, “ Candidatus Liberibacter asiaticus” (CLas). However, the mechanism of CLas colonization and growth in citrus hosts is poorly understood. In this study, we utilized the fruit pith tissue, which was able to maintain the CLas at a high abundance, as the materials for dual RNA-Seq and untargeted metabolome analysis, aiming to reveal the biological processes and phytochemical substances that are vital for CLas colonization and growth. We provided a genome-wide CLas transcriptome landscape in the fruit pith of three citrus cultivars with different tolerance and identified the important genes/pathways that contribute to CLas colonization and growth in the fruit pith. Metabolome profiling identified the key metabolites, which were mainly affected by CLas infection and influenced the population dynamic of CLas in fruit pith.

Abstract Aims Huanglongbing (citrus greening) is a plant disease putatively caused by the unculturable Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas), and it has caused severe damage to citrus plantations worldwide. There are no definitive treatments for this disease, and conventional disease control techniques have shown limited efficacy. This work presents an in silico evaluation of using specifically targeting anti-microbial peptides (STAMPs) consisting of a targeting segment and an antimicrobial segment to inhibit citrus greening by inhibiting the BamA protein of CLas, which is an outer membrane protein crucial for bacterial viability. Methods and results Initially, a set of peptides with a high affinity toward BamA protein were screened and evaluated via molecular docking and molecular dynamics simulations and were verified in vitro via bio-layer interferometry (BLI). In silico studies and BLI experiments indicated that two peptides, HASP2 and HASP3, showed stable binding to BamA. Protein structures for STAMPs were created by fusing known anti-microbial peptides (AMPs) with the selected short peptides. The binding of STAMPs to BamA was assessed using molecular docking and binding energy calculations. The attachment of high-affinity short peptides significantly reduced the free energy of binding for AMPs, suggesting that it would make it easier for the STAMPs to bind to BamA. Efficacy testing in vitro using a closely related CLas surrogate bacterium showed that STAMPs had greater inhibitory activity than AMP alone. Conclusions In silico and in vitro results indicate that the STAMPs can inhibit CLas surrogate Rhizobium grahamii more effectively compared to AMPs, suggesting that STAMPs can achieve better inhibition of CLas, potentially via enhancing the site specificity of AMPs.

Huanglongbing (HLB) is a disease caused by the phloem- limited Candidatus Liberibacter asiaticus (CLas) that affects the citrus industry worldwide. To date, only indirect strategies have been implemented to eradicate HLB. Included among these is the population control of the psyllid vector (Diaphorina citri), which usually provides inconsistent results. Even though strategies for direct CLas suppression seem a priori more promising, only a handful of reports have been focused on a confrontation of the pathogen. Recent developments in polymer chemistry have allowed the design of polycationic self-assembled block copolymers with outstanding antibacterial capabilities. Here, we report the use of polymeric nano-sized bactericide particles (PNB) to control CLas directly in the phloem vasculature. The field experiments were performed in Rioverde, San Luis Potosí, and is one of the most important citrusproducing regions in Mexico. An average 52% reduction in the bacterial population was produced when PNB was injected directly into the trunk of 20 infected trees, although, in some cases, reduction levels reached 97%. These results position PNB as a novel and promising nanotechnological tool for citrus crop protection against CLas and other related pathogens.

Abstract The Candidate Phyla Radiation (CPR) encompasses widespread uncultivated bacteria with reduced genomes and limited metabolic capacities. Most CPR bacteria lack the minimal set of enzymes required for peptidoglycan (PG) synthesis, leaving it unclear how these bacteria produce this essential envelope component. In this study, we analysed the distribution of d ‐amino acid racemases that produce the universal PG components d ‐glutamate ( d ‐Glu) or d ‐alanine ( d ‐Ala). We also examined moonlighting enzymes that synthesize d ‐Glu or d ‐Ala. Unlike other phyla in the domain Bacteria, CPR bacteria do not exhibit these moonlighting activities and have, at most, one gene encoding either a Glu or Ala racemase. One of these ‘orphan’ racemases is a predicted Glu racemase (MurI CPR ) from the CPR bacterium Candidatus Saccharimonas aalborgenesis . The expression of MurI CPR restores the growth of a Salmonella d ‐Glu auxotroph lacking its endogenous racemase and results in the substitution of l ‐Ala by serine as the first residue in a fraction of the PG stem peptides. In vitro, MurI CPR exclusively racemizes Glu as a substrate. Therefore, Ca. Saccharimonas aalborgensis may couple Glu racemization to serine and d ‐Glu incorporation into the stem peptide. Our findings provide the first insights into the synthesis of PG by an uncultivated environmental bacterium and illustrate how to experimentally test enzymatic activities from CPR bacteria related to PG metabolism.