Candidatus Liberibacter' species are insect-transmitted, phloem-limited α-Proteobacteria in the order of Rhizobiales. The citrus industry is facing significant challenges due to huanglongbing, associated with infection from 'Candidatus Liberibacter asiaticus' (Las). In order to gain greater insight into 'Ca. Liberibacter' biology and genetic diversity, we have performed genome sequencing and comparative analyses of diverse 'Ca. Liberibacter' species, including those that can infect citrus. Our phylogenetic analysis differentiates 'Ca. Liberibacter' species and Rhizobiales in separate clades and suggests stepwise evolution from a common ancestor splitting first into nonpathogenic Liberibacter crescens followed by diversification of pathogenic 'Ca. Liberibacter' species. Further analysis of Las genomes from different geographical locations revealed diversity among isolates from the United States. Our phylogenetic study also indicates multiple Las introduction events in California and spread of the pathogen from Florida to Texas. Texan Las isolates were closely related, while Florida and Asian isolates exhibited the most genetic variation. We have identified conserved Sec translocon (SEC)-dependent effectors likely involved in bacterial survival and virulence of Las and analysed their expression in their plant host (citrus) and insect vector (Diaphorina citri). Individual SEC-dependent effectors exhibited differential expression patterns between host and vector, indicating that Las uses its effector repertoire to differentially modulate diverse organisms. Collectively, this work provides insights into the evolution of 'Ca. Liberibacter' species, the introduction of Las in the United States and identifies promising Las targets for disease management. K E Y W O R D S 'Candidatus Liberibacter' sp., citrus greening disease, HLB, huanglongbing, phylogenomics, SEC effector | 717 THAPA eT Al.

Background: Many microbes used for the rapid discovery and development of metabolic pathways have sensitivities to final products and process reagents. Isopentenol (3-methyl-3-buten-1-ol), a biogasoline candidate, has an established heterologous gene pathway but is toxic to several microbial hosts. Reagents used in the pretreatment of plant biomass, such as ionic liquids, also inhibit growth of many host strains. We explored the use of Corynebacterium glutamicum as an alternative host to address these constraints. Results: We found C. glutamicum ATCC 13032 to be tolerant to both the final product, isopentenol, as well to three classes of ionic liquids. A heterologous mevalonate-based isopentenol pathway was engineered in C. glutamicum. Targeted proteomics for the heterologous pathway proteins indicated that the 3-hydroxy-3-methylglutaryl-coenzyme A reductase protein, HmgR, is a potential rate-limiting enzyme in this synthetic pathway. Isopentenol titers were improved from undetectable to 1.25 g/L by combining three approaches: media optimization; substitution of an NADH-dependent HmgR homolog from Silicibacter pomeroyi; and development of a C. glutamicum ∆poxB ∆ldhA host chassis. Conclusions: We describe the successful expression of a heterologous mevalonate-based pathway in the Grampositive industrial microorganism, C. glutamicum, for the production of the biogasoline candidate, isopentenol. We identified critical genetic factors to harness the isopentenol pathway in C. glutamicum. Further media and cultivation optimization enabled isopentenol production from sorghum biomass hydrolysates.

Bacterial response to metals can require complex regulation. We report an overlapping regulation for copper and zinc resistance genes in the denitrifying bacterium, Pseudomonas stutzeri RCH2, by three two-component regulatory proteins CopR1, CopR2 and CzcR. We conducted genome-wide evaluations to identify gene targets of two paralogous regulators, CopR1 and CopR2, annotated for copper signaling, and compared the results with the gene targets for CzcR, implicated in zinc signaling. We discovered that the CopRs and CzcR have largely common targets, and crossregulate a core set of P. stutzeri copper and zinc responsive genes. We established that this crossregulation is enabled by a conserved binding motif in the upstream regulatory regions of the target genes. The crossregulation is physiologically relevant as these regulators synergistically and antagonistically target multicopper oxidases, metal efflux and sequestration systems. CopR1 and CopR2 upregulate two cop operons encoding copper tolerance genes, while all three regulators downregulate a putative copper chaperone, Psest_1595. CzcR also upregulated the oprD gene and the CzcIABC Zn efflux system, while CopR1 and CopR2 downregulated these genes. Our study suggests that crossregulation of copper and zinc homeostasis can be advantageous, and in P. stutzeri this is enabled by shared binding motifs for multiple response regulators.

Although much is known about the responses of model plants to microbial features, we still lack an understanding of the extent of variation in immune perception across members of a plant family. In this work, we analyzed immune responses in Citrus and wild relatives, surveying 86 Rutaceae genotypes with differing leaf morphologies and disease resistances. We found that responses to microbial features vary both within and between members. Species in two subtribes, the Balsamocitrinae and Clauseninae, can recognize flagellin (flg22), cold shock protein (csp22) and chitin, including one feature from Candidatus Liberibacter species (csp22CLas), the bacterium associated with Huanglongbing. We investigated differences at the receptor level for the flagellin receptor FLAGELLIN SENSING 2 (FLS2) and the chitin receptor LYSIN MOTIF RECEPTOR KINASE 5 (LYK5) in citrus genotypes. We characterized two genetically linked FLS2 homologs from ‘Frost Lisbon’ lemon (Citrus ×limon, responsive) and ‘Washington navel’ orange (Citrus ×aurantium, non-responsive). Surprisingly, FLS2 homologs from responsive and non-responsive genotypes were expressed in Citrus and functional when transferred to a heterologous system. ‘Washington navel’ orange weakly responded to chitin, whereas ‘Tango’ mandarin (Citrus ×aurantium) exhibited a robust response. LYK5 alleles were identical or nearly identical between the two genotypes and complemented the Arabidopsis (Arabidopsis thaliana) lyk4/lyk5-2 mutant with respect to chitin perception. Collectively, our data indicate that differences in chitin and flg22 perception in these citrus genotypes are not the results of sequence polymorphisms at the receptor level. These findings shed light on the diversity of perception of microbial features and highlight genotypes capable of recognizing polymorphic pathogen features.

Corynebacterium glutamicum ATCC 13032 is an established and industrially-relevant microbial host that has been utilized for the expression of many desirable bioproducts. Tetra-methylpyrazine (TMP) is a naturally occurring alkylpyrazine with broad applications spanning fragrances to resins. We identified an engineered strain of C. glutamicum which produces 5 ​g/L TMP and separately, a strain which can co-produce both TMP and the biofuel compound isopentenol. Ionic liquids also stimulate TMP production in engineered strains. Using a fed batch-mode feeding strategy, ionic liquid stimulated strains produced 2.2 ​g/L of tetra-methylpyrazine. We show that feedback from a specific heterologous gene pathway on host physiology leads to acetoin accumulation and the production of TMP.

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Sec-delivered effector 1 (SDE1) from the Huanglongbing (HLB)-associated bacterium Candidatus Liberibacter asiaticus (CLas) was previously characterized as an inhibitor of defense-related, papain-like cysteine proteases (PLCPs) in vitro and in planta. Here we investigated the contributions of SDE1 to HLB progression. We found that SDE1 expression in the model plant Arabidopsis thaliana caused severe yellowing in mature leaves, reminiscent of both CLas-infection symptoms and accelerated leaf senescence. Induction of senescence signatures was also observed in the SDE1-expressing A. thaliana lines. These signatures were apparent in older leaves but not in seedlings, suggesting an age-associated effect. Furthermore, independent lines of transgenic Citrus paradisi (L.) Macfadyen (Duncan grapefruit) that express SDE1 exhibited hypersusceptibility to CLas. Similar to A. thaliana, transgenic citrus expressing SDE1 showed altered expression of senescence-associated genes, but only after infection with CLas. These findings suggest that SDE1 is a virulence factor that contributes to HLB progression, likely by inducing premature and/or accelerated senescence in citrus. This work provides new insight into HLB pathogenesis.

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