‘Candidatus Phytoplasma mali’ (‘Ca. P. mali’) has only one major membrane protein, the immunodominant membrane protein (Imp), which is regarded as being close to the ancestor of all phytoplasma immunodominant membrane proteins. Imp binds to actin and possibly facilitates its movement in the plant or insect host cells. However, protein sequences of Imp are quite diverse among phytoplasma species, thus resulting in difficulties in identifying conserved domains across species. In this work, we compare Imp protein sequences of ‘Ca. P. mali’ strain PM19 (Imp-PM19) with Imp of different strains of ‘Ca. P. mali’ and identify its actin-binding domain. Moreover, we show that Imp binds to the actin of apple (Malus x domestica), which is the host plant of ‘Ca. P. mali’. Using molecular and scanning force spectroscopy analysis, we find that the actin-binding domain of Imp-PM19 contains a highly positively charged amino acid cluster. Our result could allow investigating a possible correlation between Imp variants and the infectivity of the corresponding ‘Ca. P. mali’ isolates.
Persian lime (PL) is one of the most economically important citrus crops in the state of Veracruz, Mexico. However, it is affected by the presence of Huanglongbing (HLB) disease, caused by Candidatus Liberibacter asiaticus (CLas), an obligate biotrophic pathogen. Overall, PL shows a certain level of tolerance to HLB. Therefore, it is important to study the defense response mediated by salicylic acid (SA) against biotrophic pathogens in PL. Some genes with the capacity to participate in the SA response pathway, known as NtSABP, have been identified in Nicotiana tabacum, but the presence and activity of these genes in PL in response to HLB are unknown. The objective of this study was to identify homologues of SABP-like proteins in the PL transcriptome and to determine their differential expression level during CLas infection. SABP protein sequences from five different species, including N. tabacum, were used as model sequences in a tBLASTn search. A 3D model of the SABP protein was constructed and compared between N. tabacum and C. latifolia. We identified the direct homologous to each NtSABP gene in the PL using tBLASTn analysis, phylogenetic reconstruction, and tridimensional structure. Interestingly, the ClSABP1, ClSABP2, and ClSABP3 genes showed repression in CLas infected plants. There is at least one homologous to each NtSABP gene in PL. During CLas infection, these genes are somewhat suppressed.
Huanglongbing (HLB), or citrus greening, is the most destructive disease of cultivated citrus worldwide. Candidatus Liberibacter asiaticus (CLas), the putative causal agent of HLB, is transmitted by the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Liviidae). In Florida, D. citri was first reported in 1998, and CLas was confirmed in 2005. Management of HLB relies on the use of insecticides to reduce vector populations. In 2016, antibiotics were approved to manage CLas infection in citrus. Diaphorina citri is host to several bacterial endosymbionts and reducing endosymbiont abundance is known to cause a corresponding reduction in host fitness. We hypothesized that applications of oxytetracycline and streptomycin would reduce: CLas populations in young and mature citrus trees, CLas acquisition by D. citri, and D. citri abundance. Our results indicate that treatment of citrus with oxytetracycline and streptomycin reduced acquisition of CLas by D. citri adults and emerging F1 nymphs as compared with that observed in trees treated only with insecticides, but not with antibiotics. However, under field conditions, neither antibiotic treatment frequency tested affected CLas infection of young or mature trees as compared with insecticide treatment alone (negative control); whereas trees enveloped with mesh screening that excluded vectors did prevent bacterial infection (positive control). Populations of D. citri were not consistently affected by antibiotic treatment under field conditions, as compared with an insecticide only comparison. Collectively, our results suggest that while foliar application of oxytetracycline and streptomycin to citrus reduces acquisition of CLas bacteria by the vector, even high frequency applications of these formulations under field conditions do not prevent or reduce tree infection.
Comparing obligate endosymbionts with their free-living relatives is a powerful approach to investigate the evolution of symbioses, and it has led to the identification of several genomic traits consistently associated with the establishment of symbiosis. ‘Candidatus Nebulobacter yamunensis’ is an obligate bacterial endosymbiont of the ciliate Euplotes that seemingly depends on its host for survival. A subsequently characterized bacterial strain with an identical 16S rRNA gene sequence, named
, can instead be maintained in pure culture. We analysed the genomes of ‘Candidatus Nebulobacter’ and
seeking to identify key differences between their functional traits and genomic structure that might shed light on a recent transition to obligate endosymbiosis. Surprisingly, we found almost no such differences: the two genomes share a high level of sequence identity, the same overall structure, and largely overlapping sets of genes. The similarities between the genomes of the two strains are at odds with their different ecological niches, confirmed here with a parallel growth experiment. Although other pairs of closely related symbiotic/free-living bacteria have been compared in the past, ‘Candidatus Nebulobacter’ and
represent an extreme example proving that a small number of (unknown) factors might play a pivotal role in the earliest stages of obligate endosymbiosis establishment.
“Candidatus Liberibacter asiaticus” (CLas) is a phloem-restricted α-proteobacterium that is associated with citrus huanglongbing (HLB), which is the most destructive disease that affects all varieties of citrus. Although midrib is usually used as a material for CLas detection, we recently found that the bacterium was enriched in fruits, especially in the fruit pith. However, no study has revealed the molecular basis of these two parts in responding to CLas infection. Therefore, we performed transcriptome and UHPLC–MS-based targeted and untargeted metabolomics analyses in order to organize the essential genes and metabolites that are involved. Transcriptome and metabolome characterized 4834 differentially expressed genes (DEGs) and 383 differentially accumulated metabolites (DAMs) between the two materials, wherein 179 DEGs and 44 DAMs were affected by HLB in both of the tissues, involving the pathways of phenylpropanoid biosynthesis, phytohormone signaling transduction, starch and sucrose metabolism, and photosynthesis. Notably, we discovered that the gene expression that is related to beta-glucosidase and endoglucanase was up-regulated in fruits. In addition, defense-related gene expression and metabolite accumulation were significantly down-regulated in infected fruits. Taken together, the decreased amount of jasmonic acid, coupled with the reduced accumulation of phenylpropanoid and the increased proliferation of indole-3-acetic acid, salicylic acid, and abscisic acid, compared to leaf midribs, may contribute largely to the enrichment of CLas in fruit piths, resulting in disorders of photosynthesis and starch and sucrose metabolism.
In addition to Mycoplasma haemocanis and Candidatus Mycoplasma haematoparvum, a few hemoplasma species that mainly infect other livestock have been detected in dogs. ‘Candidatus Mycoplasma haemobos’ (Ca. M. haemobos) has been found in a variety of animals in China. The present study was aimed to investigate the occurrence of ‘Ca. M. haemobos’ infections in dogs and ticks collected from the Henan province, China.
Overall, 55 dog blood samples and 378 ticks on skins were collected from anemic and healthy dogs, and these samples were subjected to PCR, sequence analysis, and identification. The results showed that Haemaphysalis longicornis (266) and Rhipicephalus (Boophilus) microplus (112) were the only two parasitic ticks on dogs. Molecular detection revealed that 163 M. haemocanis, 88 ‘Ca. M. haemobos’ and 32 Anaplasma platys positive amplicons could be amplified from dogs, H. longicornis and R. (B.) microplus. In addition, co-infections (M. haemocanis + A. platys and ‘Ca. M. haemobos’+ A. platys) could be also detected.
To the best of our knowledge, this is the first molecular evidence of ‘Ca. M. haemobos’ natural infection in dogs and tick species identified as H. longicornis and R. (B.) microplus from China.
The appearance of the novel porcine haemotrophic mycoplasma (HM) species ‘Candidatus Mycoplasma haemosuis’ was reported in apparently healthy but also in clinically sick animals in China, Korea and in a case report from Germany. Outside of Asia, however, nothing further is known about the frequency of ‘Ca. M. haemosuis’ in pigs to date. To investigate the distribution of this novel HM species in Germany, fattening pigs, sows and pre-suckling piglets were examined using a herein developed quantitative real-time PCR assay (qPCR). Because the piglets were sampled before the first colostrum uptake, additional information on a possible vertical transmission from dams to their offspring was obtained.
Our novel qPCR assay successfully detected ‘Ca. M. haemosuis’ in all blood samples from the ‘Ca. M. haemosuis’-infected pigs. No cross-reactivity was detected when DNA from non-target Mycoplasma spp. and other bacterial species representing 105 bacteria/reaction were used as a template. The lower limit of detection of the qPCR was thus 10 gap gene copies per reaction and 2.5 x 103 genome equivalents (GE) per mL blood.
‘Candidatus M. haemosuis’ was detected by this qPCR in blood samples from a total out of 6.25% sows (13/208), 4.50% pre-suckling piglets (28/622) and 17.50% fattening pigs (35/200). On farm level, 3 out of 21 piglet producing farms (14.28%) and 9 out of 20 fattening farms (45.00%) were positive for ‘Ca. M. haemosuis’. Co-infections with M. suis were evident in all age groups.
‘Candidatus M. haemosuis’ infection is present in German pig farms and the detection of the novel porcine HM species in piglets immediately after birth before colostrum intake indicates vertical transmission. The novel qPCR assay specific for ‘Ca. M. haemosuis’ described herein will be a prerequisite for future studies on the prevalence, epidemiology as well as the clinical and economic impact of ‘Ca. M. haemosuis’ infections.
‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the devastating citrus ‘greening’ disease. All attempts to achieve axenic growth and complete Koch’s postulates with CLas have failed to date, at best yielding complex cocultures with very low CLas titers detectable only by PCR. Reductive genome evolution has rendered all pathogenic ‘Ca. Liberibacter’ spp. deficient in multiple key biosynthetic, metabolic and structural pathways that are highly unlikely to be rescued in vitro by media supplementation alone. By contrast, Liberibacter crescens (Lcr) is axenically cultured and its genome is both syntenic and highly similar to CLas. Our objective is to achieve replicative axenic growth of CLas via addition of missing culturability-related Lcr genes.
Bioinformatic analyses identified 405 unique ORFs in Lcr but missing (or truncated) in all 24 sequenced CLas strains. Site-directed mutagenesis confirmed and extended published EZ-Tn5 mutagenesis data, allowing elimination of 310 of these 405 genes as nonessential, leaving 95 experimentally validated Lcr genes as essential for CLas growth in axenic culture. Experimental conditions for conjugation of large GFP-expressing plasmids from Escherichia coli to Lcr were successfully established for the first time, providing a practical method for transfer of large groups of ‘essential’ Lcr genes to CLas.