Metabolomic Evaluation of Ralstonia solanacearum Cold Shock Protein Peptide (csp22)-Induced Responses in Solanum lycopersicum

Ralstonia solanacearum, the causal agent of bacterial wilt, is one of the most destructive bacterial plant pathogens. This is linked to its evolutionary adaptation to evade host surveillance during the infection process since many of the pathogen’s associated molecular patterns escape recognition. However, a 22-amino acid sequence of R. solanacearum-derived cold shock protein (csp22) was discovered to elicit an immune response in the Solanaceae. Using untargeted metabolomics, the effects of csp22-elicitation on the metabolome of Solanum lycopersicum leaves were investigated. Additionally, the study set out to discover trends that may suggest that csp22 inoculation bestows enhanced resistance on tomato against bacterial wilt. Results revealed the redirection of metabolism toward the phenylpropanoid pathway and sub-branches thereof. Compared to the host response with live bacteria, csp22 induced a subset of the discriminant metabolites, but also metabolites not induced in response to R. solanacearum. Here, a spectrum of hydroxycinnamic acids (especially ferulic acid), their conjugates and derivatives predominated as signatory biomarkers. From a metabolomics perspective, the results support claims that csp22 pre-treatment of tomato plants elicits increased resistance to R. solanacearum infection and contribute to knowledge on plant immune systems operation at an integrative level. The functional significance of these specialized compounds may thus support a heightened state of defense that can be applied to ward off attacking pathogens or toward priming of defense against future infections.

Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt’s minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

Csp1, a Cold Shock Protein Homolog in Xylella fastidiosa Influences Cell Attachment, Pili Formation, and Gene Expression

Xylella fastidiosa is a major threat to the worldwide agriculture industry (1, 2). Despite its global importance, many aspects of X. fastidiosa biology and pathogenicity are poorly understood.

Two energy barriers and a transient intermediate state determine the unfolding and folding dynamics of cold shock protein

Cold shock protein (Csp) is a typical two-state folding model protein which has been widely studied by biochemistry and single molecule techniques. Recently two-state property of Csp was confirmed by atomic force microscopy (AFM) through direct pulling measurement, while several long-lifetime intermediate states were found by force-clamp AFM. We systematically studied force-dependent folding and unfolding dynamics of Csp using magnetic tweezers with intrinsic constant force capability. Here we report that Csp mostly folds and unfolds with a single step over force range from 5 pN to 50 pN, and the unfolding rates show different force sensitivities at forces below and above ~8 pN, which determines a free energy landscape with two barriers and a transient intermediate state between them along one transition pathway. Our results provide a new insight on protein folding mechanism of two-state proteins.

Global identification of full-length cassava lncRNAs unveils the role of CRIR1 in cold stress response

Long non-coding RNAs (lncRNAs) have been considered to be important regulators of gene expression in a range of biological processes in plants. A large number of lncRNAs have been identified in plants. However, most of their biological functions still remain to be determined. Here, we identified total 3 004 lncRNAs in cassava under normal or cold-treated conditions from Iso-seq data. We further characterized a lincRNA, CRIR1, as a novel positive regulator of the plant response to cold stress. CRIR1 can be significantly induced by cold treatment. Overexpression of CRIR1 in cassava enhanced the cold tolerance of transgenic plants. Transcriptome analysis demonstrated that CRIR1 regulates a range of cold stress-related genes in a CBF-independent pathway. We further found that CRIR1 RNA can interact with MeCSP5, a homolog of the cold shock protein that acts as RNA chaperones, indicating that CRIR1 may recruit MeCSP5 to improve the translation efficiency of mRNA. In summary, our study greatly extends the repertoire of lncRNAs in plants as well as its responding to cold stress. Moreover, it reveals a sophisticated mechanism by which CRIR1 regulates plant cold stress response by modulating the expression of stress-responsive genes and increasing the translational yield.

A Cold-Shock Protein from the South Pole-Dwelling Soil Bacterium Arthrobacter sp. Confers Cold Tolerance to Rice

Low temperature is a critical environmental factor restricting the physiology of organisms across kingdoms. In prokaryotes, cold shock induces the expression of various genes and proteins involved in cellular processes. Here, a cold-shock protein (ArCspA) from the South Pole-dwelling soil bacterium Arthrobacter sp. A2-5 was introduced into rice, a monocot model plant species. Four-week-old 35S:ArCspA transgenic rice plants grown in a cold chamber at 4 °C survived for 6 days. Cold stress significantly decreased the chlorophyll content in WT plants after 4 days compared with that in 35S:ArCspA transgenic plants. RNA-seq analysis was performed on WT and 35S:ArCspA transgenic rice with/without cold stress. GO terms such as “response to stress (GO:0006950)”, “response to cold (GO:0009409)”, and “response to heat (GO:0009408)” were significantly enriched among the upregulated genes in the 35S:ArCspA transgenic rice under normal conditions, even without cold-stress treatment. The expression of five cold stress-related genes, Rab16B (Os11g0454200), Rab21 (Os11g0454300), LEA22 (Os01g0702500), ABI5 (Os01 g0859300), and MAPK5 (Os03g0285800), was significantly upregulated in the transgenic rice compared with the WT rice. These results indicate that the ArCspA gene might be involved in the induction of cold-responsive genes and provide cold tolerance.

The molecular interplay of the establishment of an infection – gene expression of Diaphorina citri gut and Candidatus Liberibacter asiaticus

Background Candidatus Liberibacter asiaticus (CLas) is one the causative agents of greening disease in citrus, an unccurable, devastating disease of citrus worldwide. CLas is vectored by Diaphorina citri, and the understanding of the molecular interplay between vector and pathogen will provide additional basis for the development and implementation of successful management strategies. We focused in the molecular interplay occurring in the gut of the vector, a major barrier for CLas invasion and colonization. Results We investigated the differential expression of vector and CLas genes by analyzing a de novo reference metatranscriptome of the gut of adult psyllids fed of CLas-infected and healthy citrus plants for 1-2, 3-4 and 5-6 days. CLas regulates the immune response of the vector affecting the production of reactive species of oxygen and nitrogen, and the production of antimicrobial peptides. Moreover, CLas overexpressed peroxiredoxin, probably in a protective manner. The major transcript involved in immune expression was related to melanization, a CLIP-domain serine protease we believe participates in the wounding of epithelial cells damaged during infection, which is supported by the down-regulation of pangolin. We also detected that CLas modulates the gut peristalsis of psyllids through the down-regulation of titin, reducing the elimination of CLas with faeces. The up-regulation of the neuromodulator arylalkylamine N-acetyltransferase implies CLas also interferes with the double brain-gut communication circuitry of the vector. CLas colonizes the gut by expressing two Type IVb pilin flp genes and several chaperones that can also function as adhesins. We hypothesized biofilm formation occurs by the expression of the cold shock protein of CLas. Conclusions The thorough detailed analysis of the transcritome of Ca. L. asiaticus and of D. citri at different time points of their interaction in the gut tissues of the host led to the identification of several host genes targeted for regulation by L. asiaticus, but also bacterial genes coding for potential effector proteins. The identified targets and effector proteins are potential targets for the development of new management strategies directed to interfere with the successful utilization of the psyllid vector by this pathogen.

Impact of crowded environments on binding between protein and single-stranded DNA

The concept of Molecular Crowding depicts the high density of diverse molecules present in the cellular interior. Here, we determine the impact of low molecular weight and larger molecules on binding capacity of single-stranded DNA (ssDNA) to the cold shock protein B (CspB). Whereas structural features of ssDNA-bound CspB are fully conserved in crowded environments as probed by high-resolution NMR spectroscopy, intrinsic fluorescence quenching experiments reveal subtle changes in equilibrium affinity. Kinetic stopped-flow data showed that DNA-to-protein association is significantly retarded independent of choice of the molecule that is added to the solution, but dissociation depends in a nontrivial way on its size and chemical characteristics. Thus, for this DNA–protein interaction, excluded volume effect does not play the dominant role but instead observed effects are dictated by the chemical properties of the crowder. We propose that surrounding molecules are capable of specific modification of the protein’s hydration shell via soft interactions that, in turn, tune protein–ligand binding dynamics and affinity.

Csp1, A Cold-Shock Protein Homolog in Xylella fastidiosa Influences Pili Formation, Stress Response, and Gene Expression

Bacterial cold shock-domain proteins (CSPs) are conserved nucleic acid binding chaperones that play important roles in stress adaptation and pathogenesis. Csp1 is a temperature-independent cold shock protein homolog in Xylella fastidiosa, a bacterial plant pathogen of grapevine and other economically important crops. Csp1 contributes to stress tolerance and virulence in X. fastidiosa. However, besides general single stranded nucleic acid binding activity, little is known about the specific function(s) of this protein. To further investigate the role(s) of Csp1, we compared phenotypic differences between wild type and a csp1 deletion mutant (Δcsp1). We observed decreases in cellular aggregation and surface attachment with the Δcsp1 strain compared to the wild type. Transmission electron microscopy imaging revealed that Δcsp1 had reduced pili compared to the wild type and complemented strains. The Δcsp1 strain also showed reduced survival after long term growth, in vitro. Since Csp1 binds DNA and RNA, its influence on gene expression was also investigated. Long-read Nanopore RNA-Seq analysis of wild type and Δcsp1 revealed changes in expression of several genes important for attachment and biofilm formation in Δcsp1. One gene of intertest,pilA1, encodes a type IV pili subunit protein and was up regulated in Δcsp1. Deleting pilA1 increased surface attachment in vitro and reduced virulence in grapevines.X. fastidiosa virulence depends on bacterial attachment to host tissue and movement within and between xylem vessels. Our results show Csp1 may play a role in both virulence and stress tolerance by influencing expression of genes important for biofilm formation.