Thermoresponsive C22 phage stiffness modulates the phage infectivity.

Bacteriophages offer a sustainable alternative for controlling crop disease. However, the lack of knowledge on phage infection mechanisms makes phage-based biological control varying and ineffective. In this work, we interrogated the temperature dependence of the infection and thermo-responsive behavior of the C22 phage. This soilborne podovirus is capable of lysing Ralstonia solanacearum, causing bacterial wilt disease. We revealed that the C22 phage could better infect the pathogenic host cell when incubated at low temperatures (25, 30 °C) than at high temperatures (35, 40 °C). Measurement of the C22 phage stiffness revealed that the phage stiffness at low temperatures was 2-3 times larger than at high temperatures. In addition, the imaging results showed that more C22 phage particles were attached to the cell surface at low temperatures than at high temperatures, associating the phage stiffness and the phage attachment. The result suggests that the structure and stiffness modulation in response to temperature change improve infection, providing mechanistic insight into the C22 phage lytic cycle. Our study signifies the need to understand phage responses to the fluctuating environment for effective phage-based biocontrol implementation.

C22 podovirus infectivity is associated with intermediate stiffness.

Bacteriophages have potential for use as biological control agents (biocontrols) of pathogenic bacteria, but their low stability is limiting for their utilization as biocontrols. Understanding of the conditions conducive to storage of phages in which infectivity is maintained over long periods will be useful for their application as biocontrols. We employed a nanomechanical approach to study how external environmental factors affect surface properties and infectivity of the podovirus C22 phage, a candidate for biocontrol of Ralstonia solanacearum, the agent of bacterial wilt in crops. We performed atomic force microscopy (AFM)-based nano-indentation on the C22 phage in buffers with varying pH and ionic strength. The infectivity data from plaque assay in the same conditions revealed that an intermediate range of stiffness was associated with phage titer that remained consistently high, even after prolonged storage up to 182 days. The data are consistent with the model that C22 phage must adopt a metastable state for maximal infectivity, and external factors that alter the stiffness of the phage capsid lead to perturbation of this infective state.

Development and application of triple antibody sandwich enzyme-linked immunosorbent assays for begomovirus detection using monoclonal antibodies against Tomato yellow leaf curl Thailand virus.

BACKGROUND: Tomato yellow leaf curl Thailand virus, TYLCTHV, is a begomovirus that causes severe losses of tomato crops in Thailand as well as several countries in Southeast and East Asia. The development of monoclonal antibodies (MAbs) and serological methods for detecting TYLCTHV is essential for epidemiological studies and screening for virus-resistant cultivars. METHODS: The recombinant coat protein (CP) of TYLCTHV was expressed in Escherichia coli and used to generate MAbs against TYLCTHV through hybridoma technology. The MAbs were characterized and optimized to develop triple antibody sandwich enzyme-linked immunosorbent assays (TAS-ELISAs) for begomovirus detection. The efficiency of TAS-ELISAs for begomovirus detection was evaluated with tomato, pepper, eggplant, okra and cucurbit plants collected from several provinces in Thailand. Molecular identification of begomoviruses in these samples was also performed through PCR and DNA sequence analysis of the CP gene. RESULTS: Two MAbs (M1 and D2) were generated and used to develop TAS-ELISAs for begomovirus detection. The results of begomovirus detection in 147 field samples indicated that MAb M1 reacted with 2 begomovirus species, TYLCTHV and Tobacco leaf curl Yunnan virus (TbLCYnV), whereas MAb D2 reacted with 4 begomovirus species, TYLCTHV, TbLCYnV, Tomato leaf curl New Delhi virus (ToLCNDV) and Squash leaf curl China virus (SLCCNV). Phylogenetic analyses of CP amino acid sequences from these begomoviruses revealed that the CP sequences of begomoviruses recognized by the narrow-spectrum MAb M1 were highly conserved, sharing 93% identity with each other but only 72-81% identity with MAb M1-negative begomoviruses. The CP sequences of begomoviruses recognized by the broad-spectrum MAb D2 demonstrated a wider range of amino acid sequence identity, sharing 78-96% identity with each other and 72-91% identity with those that were not detected by MAb D2. CONCLUSIONS: TAS-ELISAs using the narrow-specificity MAb M1 proved highly efficient for the detection of TYLCTHV and TbLCYnV, whereas TAS-ELISAs using the broad-specificity MAb D2 were highly efficient for the detection of TYLCTHV, TbLCYnV, ToLCNDV and SLCCNV. Both newly developed assays allow for sensitive, inexpensive, high-throughput detection of begomoviruses in field plant samples, as well as screening for virus-resistant cultivars.

Two asian jumbo phages, ϕRSL2 and ϕRSF1, infect Ralstonia solanacearum and show common features of ϕKZ-related phages.

Jumbo phages infecting Ralstonia solanacearum were isolated in Thailand (ϕRSL2) and Japan (ϕRSF1). They were similar regarding virion morphology, genomic arrangement, and host range. Phylogenetic and proteomic tree analyses demonstrate that the ϕRSL2 and ϕRSF1 belong to a group of evolutionary related phages, including Pseudomonas phages ϕKZ, 201ϕ2-1 and all previously described ϕKZ-related phages. Despite conserved genomic co-linearity between the ϕRSL2 and ϕRSF1, they differ in protein separation patterns. A major difference was seen in the detection of virion-associated-RNA polymerase subunits. All ß- and ß'-subunits were detected in ϕRSF1, but one ß'-subunit was undetected in ϕRSL2. Furthermore, ϕRSF1 infected host cells faster (latent period: 60 and 150min for ϕRSF1 and ϕRSL2, respectively) and more efficiently than ϕRSL2. Therefore, the difference in virion-associated-RNA polymerase may affect infection efficiency. Finally, we show that ϕRSF1 is able to inhibit bacterial wilt progression in tomato plants.

Genomic diversity of large-plaque-forming podoviruses infecting the phytopathogen Ralstonia solanacearum.

The genome organization, gene structure, and host range of five podoviruses that infect Ralstonia solanacearum, the causative agent of bacterial wilt disease were characterized. The phages fell into two distinctive groups based on the genome position of the RNA polymerase gene (i.e., T7-type and ϕKMV-type). One-step growth experiments revealed that ϕRSB2 (a T7-like phage) lysed host cells more efficiently with a shorter infection cycle (ca. 60 min corresponding to half the doubling time of the host) than ϕKMV-like phages such as ϕRSB1 (with an infection cycle of ca. 180 min). Co-infection experiments with ϕRSB1 and ϕRSB2 showed that ϕRSB2 always predominated in the phage progeny independent of host strains. Most phages had wide host-ranges and the phage particles usually did not attach to the resistant strains; when occasionally some did, the phage genome was injected into the resistant strain's cytoplasm, as revealed by fluorescence microscopy with SYBR Gold-labeled phage particles.