Search

Having bacteria grown in pure culture has been the foundation of bacteriology, by allowing a wide range of microbiological studies towards understanding the functionality of a specific bacterium. However, most bacteria have not been axenically cultured to date, thus hindering the understanding of their role in the context of their host or environment. One of these unculturable bacteria are the recently emergent plant pathogens ‘Candidatus Liberibacter spp.’. This group is comprised by dynamic psyllid-vectored, phloem-limited plant pathogens and endophytes that harm a wide range of economically important crops worldwide. ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with Huanglongbing (HLB) in most of the main citrus-producing areas globally, a disease causing severe economic damages. Although the establishment of axenic cultures of CLas remains a major scientific challenge, many research groups have devoted efforts to culture this bacterium to aid in elucidating its virulence mechanism and contribute towards effective HLB management. This led to the development of innovative systems to culture and grow CLas, however different authors have approached the concepts of bacterial culture and axenic culture in different manners, leading to confusion in the terminology used. In this review, we provide the scientific definitions of important terms in bacteriology, while critically reviewing the contribution of each of these important CLas culturing studies.

Cellular responses to biotic stress frequently involve signaling pathways that are conserved across eukaryotes. These pathways include the cytoskeleton, a proteinaceous network that senses external cues at the cell surface and signals to interior cellular components. During biotic stress, dynamic cytoskeletal rearrangements serve as a platform from which early immune-associated processes are organized and activated. Bacterial pathogens of plants and animals use proteins called type III effectors (T3Es) to interfere with host immune signaling, thereby promoting virulence. We previously found that RipU, a T3E from the soilborne phytobacterial pathogen Ralstonia solanacearum, co-localizes with the plant cytoskeleton. Here, we show that RipU from R. solanacearum K60 (RipUK60) associated with and altered the organization of both the actin and microtubule cytoskeleton. We found that pharmacological disruption of the tomato (Solanum lycopersicum) cytoskeleton promoted R. solanacearum K60 colonization. Importantly, tomato plants inoculated with R. solanacearum K60 lacking RipUK60 (ΔripUK60) had reduced wilting symptoms and significantly reduced root colonization when compared to plants inoculated with wild-type R. solanacearum K60. Collectively, our data suggest that R. solanacearum K60 uses the type III effector RipUK60 to remodel cytoskeletal organization, thereby promoting pathogen virulence. Author summary: Pathogenic bacteria secrete dozens of proteins directly into plant and animal cells to interfere with host biology and promote disease. These proteins, termed effectors, target different host proteins to alter cellular processes and enable pathogen virulence. Understanding how distinct effectors manipulate host proteins is critical for developing new disease control strategies. The cytoskeleton is a protein network found across eukaryotic organisms that can sense external signals and transmit responses to the cell interior. Dynamic reorganization of the cytoskeleton is required for immune signaling. Here we showed that the cytoskeleton is critical for resistance to the soilborne phytobacterial pathogen Ralstonia solanacearum. We found that RipU, an R. solanacearum effector protein, physically associates with and alters the organization of the actin and microtubule cytoskeleton in plants. We also found that RipU is required for full pathogen virulence. While an R. solanacearum mutant that is defective in ripU is unable to cause disease at the same level as wild type R. solanacearum, chemical disruption of the cytoskeleton restored full virulence to these mutants. Our study provides new insights into how pathogenic bacteria manipulate cellular targets to cause disease. [ABSTRACT FROM AUTHOR]

Xylella fastidiosa is a plant pathogenic bacterium with devastating consequences to several crops of economic importance across the world. While this pathogen has been studied for over a century in the United States, several aspects of its biology remain to be investigated. Determining the physiological state of bacteria is essential to understand the effects of its interactions with different biotic and abiotic factors on cell viability. Although X. fastidiosa is culturable, its slow growing nature makes this technique cumbersome to assess the physiological state of cells present in a given environment. PMA-qPCR, i.e. the use of quantitative PCR combined with the pre-treatment of cells with the dye propidium monoazide, has been successfully used in a number of studies on human pathogens to calculate the proportion of viable cells, but has less frequently been tested on plant pathogens. We found that the use of a version of PMA, PMAxx, facilitated distinguishing between viable and non-viable cells based on cell membrane integrity in vitro and in planta. Additional experiments comparing the number of culturable, viable, and total cells in planta would help further confirm our initial results. Enhancers, intended to improve the efficacy of PMAxx, were not effective and appeared to be slightly toxic to X. fastidiosa.

A group of aggressive pathogens have evolved to colonize the plant xylem. In this vascular tissue, where water and nutrients are transported from the roots to the rest of the plant, pathogens must be able to thrive under acropetal xylem sap flow and scarcity of nutrients while having direct contact only with predominantly dead cells. Nevertheless, a few bacteria have adapted to exclusively live in the xylem, and various pathogens may colonize other plant niches without causing symptoms unless they reach the xylem. Once established, the pathogens modulate its physicochemical conditions to enhance their growth and virulence. Adaptation to the restrictive lifestyle of the xylem leads to genome reduction in xylem-restricted bacteria, as they have a higher proportion of pseudogenes in their genome. The basis of xylem adaptation is not completely understood; therefore, a need still exists for model systems to advance the knowledge on this topic. [ABSTRACT FROM AUTHOR]

The phloem-restricted, insect-transmitted bacterium ‘Candidatus Liberibacter asiaticus’ (CLas) is associated with huanglongbing (HLB), the most devastating disease of citrus worldwide. The inability to culture CLas impairs the understanding of its virulence mechanisms and the development of effective management strategies to control this incurable disease. Previously, our research group used commercial grapefruit juice (GJ) to prolong the viability of CLas in vitro. In the present study, GJ was amended with a wide range of compounds and incubated under different conditions to optimize CLas growth. Remarkably, results showed that CLas growth ratios were inversely proportional to the initial inoculum concentration. This correlation is probably regulated by a cell density-dependent mechanism, because diluting samples between subcultures allowed CLas to resume growth. Moreover, strategies to reduce the cell density of CLas, such as subculturing at short intervals and incubating samples under flow conditions, allowed this bacterium to multiply and reach maximum growth as early as 3 days after inoculation, although no sustained exponential growth was observed under any tested condition. Unfortunately, cultures were only transient, because CLas lost viability over time; nevertheless, we obtained populations of about 105 genome equivalents/ml repeatedly. Finally, we established an ex vivo system to grow CLas within periwinkle calli that could be used to propagate bacterial inoculum in the lab. In this study we determined the influence of a comprehensive set of conditions and compounds on CLas growth in culture. We hope our results will help guide future efforts toward the long-sought goal of culturing CLas axenically. [ABSTRACT FROM AUTHOR]

A wide range of potentially plant pathogenic microorganisms are naturally present in the environment. Despite relying only on the innate immune system, plants are able to resist most of the pathogens. Plants employ a multi‐layered defence system in which the first layer triggers the basal resistance (pathogen‐associated molecular pattern‐triggered immunity [PTI]). The second layer occurs when a resistance protein (R protein) that mostly encodes nucleotide‐binding leucine‐rich repeat receptors (NLRs) recognises an effector molecule secreted by an adapted pathogen, leading to effector‐triggered immunity (ETI), which triggers the hypersensitive response (HR). More recently, ETI was shown to restore and potentiate PTI signalling components, leading to a robust immune response. Multiple mechanisms of regulation are employed to guarantee proper HR activation. NLR proteins can interact between them and form a heel‐like pentamer that anchors to the plasma membrane. Furthermore, NLRs and other proteins can cooperate with NLRs to propagate the immune signalling. Downstream to the recognition of the pathogen by the plant, a rapid cellular response is initiated involving the generation of signalling events that precedes the HR. Here, we summarise the mechanisms involved in HR and highlight new advances in the knowledge of the immune system signalling. We also approach the role of HR threshold during infection by biotrophic, necrotrophic and hemibiotrophic pathogens and the impact in plant fitness and the community of pathogens found in the environment. [ABSTRACT FROM AUTHOR]

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown. [ABSTRACT FROM AUTHOR]