Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host.

The course of microbial infection in insects is shaped by a two-stage process of immune defense. Constitutive defenses, such as engulfment and melanization, act immediately and are followed by inducible defenses, archetypically the production of antimicrobial peptides, which eliminate or suppress the remaining microbes. By applying RNAseq across a 7-day time course, we sought to characterize the long-lasting immune response to bacterial challenge in the mealworm beetle Tenebrio molitor, a model for the biochemistry of insect immunity and persistent bacterial infection. By annotating a hybrid de novo assembly of RNAseq data, we were able to identify putative orthologs for the majority of components of the conserved insect immune system. Compared with Tribolium castaneum, the most closely related species with a reference genome sequence and a manually curated immune system annotation, the T. molitor immune gene count was lower, with lineage-specific expansions of genes encoding serine proteases and their countervailing inhibitors accounting for the majority of the deficit. Quantitative mapping of RNAseq reads to the reference assembly showed that expression of genes with predicted functions in cellular immunity, wound healing, melanization, and the production of reactive oxygen species was transiently induced immediately after immune challenge. In contrast, expression of genes encoding antimicrobial peptides or components of the Toll signaling pathway and iron sequestration response remained elevated for at least 7 days. Numerous genes involved in metabolism and nutrient storage were repressed, indicating a possible cost of immune induction. Strikingly, the expression of almost all antibacterial peptides followed the same pattern of long-lasting induction, regardless of their spectra of activity, signaling possible interactive roles in vivo.

Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes whereas antibiotics are mostly employed by microorganisms. As antimicrobial peptides (AMPs) mostly target the cell wall, a microbial ‘Achilles heel’, it has been proposed that bacterial resistance evolution is very unlikely and hence AMPs are ancient ‘weapons’ of multicellular organisms. Here we provide a new hypothesis to explain the widespread distribution of AMPs amongst multicellular organism. Studying five antimicrobial peptides from vertebrates and insects, we show, using a classic Luria-Delbrück fluctuation assay, that cationic antimicrobial peptides (AMPs) do not increase bacterial mutation rates. Moreover, using rtPCR and disc diffusion assays we find that AMPs do not elicit SOS or rpoS bacterial stress pathways. This is in contrast to the main classes of antibiotics that elevate mutagenesis via eliciting the SOS and rpoS pathways. The notion of the ‘Achilles heel’ has been challenged by experimental selection for AMP-resistance, but our findings offer a new perspective on the evolutionary success of AMPs. Employing AMPs seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs. Cationic antimicrobial peptides are ancient and ubiquitous immune effectors that multicellular organisms use to kill and police microbes, whilst antibiotics are mostly employed by microorganisms. Here we provide a new hypothesis to explain this widespread adoption of antimicrobial peptides. We show that cationic antimicrobial peptides (AMPs) do not increase bacterial mutagenesis, as they do not elicit bacterial stress pathways. Those stress pathways increase the mutation rate when bacteria are treated with antibiotics. Employing AMPs hence seems advantageous for multicellular organisms, as it does not fuel the adaptation of bacteria to their immune defenses. This has important consequences for our understanding of host-microbe interactions, the evolution of innate immune defenses, and also sheds new light on antimicrobial resistance evolution and the use of AMPs as drugs.

Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.

Insects show long-lasting antimicrobial immune responses that follow the initial fast-acting cellular processes. These immune responses are discussed to provide a form of phrophylaxis and/or to serve as a safety measure against persisting infections. The duration and components of such long-lasting responses have rarely been studied in detail, a necessary prerequisite to understand their adaptive value. Here, we present a 21 day proteomic time course of the mealworm beetle Tenebrio molitor immune-challenged with heat-killed Staphylococcus aureus . The most upregulated peptides are antimicrobial peptides (AMPs), many of which are still highly abundant 21 days after infection. The identified AMPs included toll and imd-mediated AMPs, a significant number of which have no known function against S. aureus or other Gram-positive bacteria. The proteome reflects the selective arena for bacterial infections. The results also corroborate the notion of synergistic interactions in vivo that are difficult to model in vitro . This article is part of the themed issue ‘Evolutionary ecology of arthropod antimicrobial peptides’.

Pseudomonas aeruginosa is the causative agent of chronic respiratory infections and is an important pathogen of cystic fibrosis patients. Adaptive mutations play an essential role for antimicrobial resistance and persistence. The factors that contribute to bacterial mutagenesis in this environment are not clear. Recently it has been proposed that cationic antimicrobial peptides such as LL-37 could act as mutagens in P. aeruginosa. Here we provide experimental evidence that mutagenesis is the product of a joint action of LL-37 and free iron. By estimating mutation rate, mutant frequencies and assessing mutational spectra in P. aeruginosa treated either with LL-37, iron or a combination of both we demonstrate that mutation rate and mutant frequency were increased only when free iron and LL-37 were present simultaneously. Colistin had the same effect. The addition of an iron chelator completely abolished this mutagenic effect, suggesting that LL-37 enables iron to enter the cells resulting in DNA damage by Fenton reactions. This was also supported by the observation that the mutational spectrum of the bacteria under LL-37-iron regime showed one of the characteristic Fenton reaction fingerprints: C to T transitions. Free iron concentration in nature and within hosts is kept at a very low level, but the situation in infected lungs of cystic fibrosis patients is different. Intermittent bleeding and damage to the epithelial cells in lungs may contribute to the release of free iron that in turn leads to generation of reactive oxygen species and deterioration of the respiratory tract, making it more susceptible to the infection. Cationic antimicrobial peptides (cAMPs) are small proteins naturally produced by the immune system to limit bacterial growth mainly through pore formation in the membrane. It has recently been suggested that sub-inhibitory concentrations of cAMPs promote bacterial mutagenesis, similarly to antibiotics. However, we previously reported that cAMPs do not increase mutation rate and do not activate bacterial stress responses. Here we resolve this contradiction. We report that free iron in the culture medium increases mutagenesis in the presence of cAMPs. We show that sub-inhibitory concentrations of cAMPs facilitate entry of free iron into bacterial cells, where it interacts with hydrogen peroxide, thereby resulting in production of DNA-damaging reactive oxygen species and increased mutagenesis. Moreover, these results may have clinically-relevant implications: while very little free iron is normally present in healthy individuals, this is not the case in patients suffering from cystic fibrosis, where elevated bacterial mutagenesis promotes antibiotic resistance and contributes to persistence and severity of infection. Thus, an intervention aimed at reduction of free iron in the lungs could reduce the cAMPs-facilitation of iron-mediated mutagenesis; hence antibiotic resistance and pathoadaptation.