Publications

4475

Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with endosymbioses in aerobic ciliates.

AbstractIn Argentina, amaranth is a promising crop due to high nutritional quality and ability to grow in a diversity of environments. In areas cultivated with amaranth, were observed plants exhibiting slow growth, deformed leaves, proliferation of shoots and malformed lateral panicles. Field survey revealed up to 96% disease incidence and 92% of the seeds collected from mother plants produced diseased seedlings. A phytoplasma was detected in association with seedlings and adult plants using nested PCR assays. Molecular identification by computer‐simulated RFLP and phylogenetic analysis evidenced the occurrence of a ‘Candidatus Phytoplasma hispanicum’‐related strain, affiliated with 16SrXIII‐A subgroup. The findings implicate amaranth as a new host for this subgroup and as a potential reservoir of the pathogen for other cultivated species. In addition, to the best of our knowledge, this study reports for the first time the presence of 16SrXIII‐A phytoplasma in Argentina and in South America. Furthermore, transmission assays pointed that naturally infected seed is an important vehicle of dissemination of the pathogen, threatening the expansion of the crop for new geographical areas.

AbstractBackgroundThe symbiosis between the olive fruit fly,Bactrocera oleae, andCandidatusErwinia dacicola has been demonstrated as essential for the fly’s larval development and adult physiology. The mass rearing of the olive fruit fly has been hindered by several issues, including problems which could be related to the lack of the symbiont, presumably due to preservatives and antibiotics currently used during rearing under laboratory conditions. To better understand the mechanisms underlying symbiont removal or loss during the rearing of lab colonies of the olive fruit fly, we performed experiments that focused on bacterial transfer from wild female flies to their eggs. In this research, eggs laid by wild females were treated with propionic acid solution, which is often used as an antifungal agent, a mixture of sodium hypochlorite and Triton X, or water (as a control). The presence of the bacterial symbiont on eggs was evaluated by real-time PCR and scanning electron microscopy.ResultsDGGE analysis showed a clear band with the same migration behavior present in all DGGE profiles but with a decreasing intensity. Molecular analyses performed by real-time PCR showed a significant reduction inCa. E. dacicola abundance in eggs treated with propionic acid solution or a mixture of sodium hypochlorite and Triton X compared to those treated with water. In addition, the removal of bacteria from the surfaces of treated eggs was highlighted by scanning electron microscopy.ConclusionsThe results clearly indicate how the first phases of the colony-establishment process are important in maintaining the symbiont load in laboratory populations and suggest that the use of products with antimicrobial activity should be avoided. The results also suggest that alternative rearing procedures for the olive fruit fly should be investigated.

AbstractIn order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma.

‘Candidatus Liberibacter asiaticus’ (CLas) is one of the causal agents of citrus Huanglongbing (HLB), a bacterial disease of citrus trees that greatly reduces fruit yield and quality. CLas strains produce an array of currently uncharacterized Sec-dependent secretory proteins. In this study, the conserved chromosomally encoded protein CLIBASIA_03875 was identified as a novel Sec-dependent secreted protein. We show that CLIBASIA_03875 contains a putative Sec- secretion signal peptide (SP), a 29 amino acid residue located at the N-terminus, with a mature protein (m3875) of 22 amino acids found to localize in multiple subcellular components of the leaf epidermal cells of Nicotiana benthamiana. When overexpressed via a Potato virus X (PVX)-based expression vector in N. benthamiana, m3875 suppressed programmed cell death (PCD) and the H2O2 accumulation triggered by the pro-apoptotic mouse protein BAX and the Phytophthora infestans elicitin INF1. Overexpression also resulted in a phenotype of dwarfing, leaf deformation and mosaics, suggesting that m3875 has roles in plant immune response, growth, and development. Substitution mutagenesis of the charged amino acid (D7, R9, R11, and K22) with alanine within m3875 did not recover the phenotypes for PCD and normal growth. In addition, the transiently overexpressed m3875 regulated the transcriptional levels of N. benthamiana orthologs of CNGCs (cyclic nucleotide-gated channels), BI-1 (Bax-inhibitor 1), and WRKY33 that are involved in plant defense mechanisms. To our knowledge, m3875 is the first PCD suppressor identified from CLas. Studying the function of this protein provides insight as to how CLas attenuates the host immune responses to proliferate and cause Huanglongbing disease in citrus plants.

Disease caused by the bacterial pathogen “Candidatus Liberibacter solanacearum” (Lso) represents a serious threat to solanaceous crop production. Insecticide applications to control the psyllid vector, Bactericera cockerelli Šulc (Hemiptera: Triozidae) has led to the emergence of resistance in psyllids populations. Efforts to select natural resistant cultivars have been marginally successful and have been complicated by the presence of distinct Lso haplotypes (LsoA, LsoB) differing in symptoms severity on potato and tomato. A potentially promising management tool is to boost host resistance to the pathogen and/or the insect vector by promoting mycorrhization. Here we tested the hypothesis that mycorrhizal fungi can mitigate the effect of Lso infection on tomato plants. The presence of mycorrhizal fungi substantially delayed and reduced the incidence of Lso-induced symptoms on tomato as compared to non-mycorrhized plants. However, PCR with specific Lso primers revealed that mycorrhization did not prevent Lso transmission or translocation to newly formed leaves. Mycorrhization significantly reduced oviposition by psyllids harboring LsoA and survival of nymphs from these eggs. However, mycorrhization had no effect on oviposition by psyllids harboring LsoB or the survival of nymphs from parents harboring LsoB. These findings indicate the use of mycorrhizal fungi is a promising strategy for the mitigation of disease caused by both LsoA and LsoB and warrants additional field testing.