Laser microdissection: A promising tool for exploring microorganisms and their interactions with hosts

Podgorny, Oleg V.; Лазарев, В. Н.

doi:10.1016/j.mimet.2016.01.001

Cited by 17 publications

(13 citation statements)

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“…This report is the one of the few presentation on the whole transcriptome (including mRNA, lncRNA, and circRNA) of microbes (62–64). In the classical RNA-seq work after single-cell level LCM, the global expression of mRNA by using oligodT primers is generally applied (11, 16, 17), while, for revealing the whole transcriptome, we used the DNA/RNA chimeric primer mix and SPIA ® Amplification (14, 65), and successfully acquired the mRNA, lncRNA and circRNA. However, it is imperfect that the miRNA of C.heterostrophus race O could not be sequenced in an one-off workflow.…”

Section: Discussionmentioning

confidence: 99%

“…mRNA-sequencing is the frequently used way after LCM(16, 17), while increasing reports have disclosed that noncodingRNAs play numerous roles in eucaryon (18–20), and regulate pathogen-host interaction(21–23). For example, miR393 is a conserved microRNA triggered by PAMPs, which represses F-box auxin receptors and consequently impedes auxin signaling(24).…”

Section: Introductionmentioning

confidence: 99%

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Single-cell level transcriptome of the maize pathogenic fungicochliobolus heterostrophusrace O in infection reveal the virulence related genes, and potential circRNA effector

Chen

Wang

et al. 2018

Preprint

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. CC-BY 4.0 International license It is made available under a (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/350785 doi: bioRxiv preprint first posted online 17 Abstract 18Cochliobolus heterostrophus is a crucial pathogenic fungus that causes southern 19 corn leaf blight (SCLB) in maize worldwide, however, the virulence mechanism of 20 the dominant race O remains unclear. In this report, the single-cell level of pathogen 21 tissue at three infection stages were collected from the host interaction-situ, and were 33 the pathogenic circRNA and host miRNA was predicted. An important exon-intron 34 circRNA Che-cirC2410 combines informatics characteristics above, and highly 35 expressed in the DY strain. Che-cirC2410 initiate from the pseudogene chhtt, which 36 doesn`t translate genetic code into protein. In-situ hybridization tells the sub-cellular 37 localization of Che-cirC2410 include pathogen`s mycelium， periplasm, and the 38 diseased host tissues. The target of Che-cirC2410 was predicted to be zma-miR399e-39 5P, and the interaction between noncoding RNAs was proved. More, the expression 40 of zma-miR399e-5P exhibited a negative correlation to Che-cirC2410 in vivo. The 41 deficiency of Che-circ2410 decreased the race O virulence. The host resistance to 42 SCLB was weakened when zma-miR399e-5P was silenced. Thus, a novel circRNA-43 type effector and its resistance related miRNA target are proposed cautiously in this 44 report. These findings enriched the pathogen-host dialogue by using noncoding (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/350785 doi: bioRxiv preprint first posted online 50 heterostrophus) is not optimistic in Asia, however we have limit knowledge about the 51 infection mechanism of the dominant C.heterostrophus race O. We take full 52 advantage of the ideal C.heterostrophus genome database, laser capture 53 microdissection and single-cell level RNA sequencing. Hence, we could avert the 54 artificial influence such as medium, and profile the real gene mobilization strategy in 55 the infection. The results of coding RNA section were accessible, virulence related 56 genes (such as the signal transduction, PKS, SSP) were detected in RNA-seq,which 57 accord with previous reports. However, the results of noncoding RNA was astonished, 58 2279 circular RNAs (circRNA) and 169 long noncoding RNAs (lncRNA) were 59 revealed in our results. Generally, the function of noncoding RNA was hypothesized 60 in single species, but we boldly guess that the function of circRNA is rather 61 complicated in the pathogen-host interaction. Finally, the circRNA in-situ 62 hybridization (ISH) demonstrate the secretion of pathogen circRNA into the host 63 tissue. By bioinformatic prediction, we found a sole microRNA target, and prove...

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Single-cell level transcriptome of the maize pathogenic fungicochliobolus heterostrophusrace O in infection reveal the virulence related genes, and potential circRNA effector

Chen

Wang

et al. 2018

Preprint

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“…Although the functional prerequisites of the LCM system impact negatively on microscope image quality, e.g. because samples have to be viewed through the glass slide and the optically heterogenous PEN membrane [19], we found that single OBs could be detected reliably and selected for excision when using either the ×63 or ×100 objectives.…”

Section: Full-textmentioning

confidence: 99%

“…The SOBMR may be underestimated if there is damage caused to some of the OBs during the excision or capture processes. However, the risk of OB damage is negligible because the LCM process occurs without heat transfer, the optical absorbance of nucleic acids and proteins is low in the UV-A range, and during catapulting the single laser pulse is directed not to the centre of the ROI but to the connecting bridge between the ROI and rest of the PEN membrane [19,25]. To minimize the risk of damage to OBs, the laser cutting path, which is digitally demarcated (pre-defined) on the screen, was not placed close to the target OBs.…”

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confidence: 99%

Determination of the virulence of single nucleopolyhedrovirus occlusion bodies using a novel laser capture microdissection method

Munsamy

Bouwer

2020

Journal of General Virology

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Determination of the virulence of occlusion bodies (OBs), which are the horizontal transmission structures of nucleopolyhedroviruses (NPVs), is an important area of baculovirology. A method for inoculating an insect with an isolated OB was developed using Helicoverpa armigera nucleopolyhedrovirus (HearNPV) infection of second instar Helicoverpa armigera larvae as a model NPV–host pathosystem. In this novel method, laser capture microdissection (LCM) was used to directly catapult single OBs onto the surface of insect diet in bioassay containers. Since exposure via the natural oral horizontal transmission route of each larva to a single OB was established and not subject to chance variation, the method facilitated determination of the insect mortality rate (4.8%) associated with exposure to single HearNPV OBs. Droplet feeding bioassays confirmed that the novel method did not reduce OB virulence. The LCM method sets a foundation for virulence and genetic diversity studies based on single NPV OBs.

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“…Laser capture microdissection coupled with an expression profiling technique may become a valuable tool for understanding the temporal and spatial dynamics of plant-pathogen interactions (Podgorny and Lazarev, 2016). Laser capture microdissection was applied to stomatal and surrounding areas of grapevine leaves in contact with the downy mildew pathogen Plasmopara viticola during early stages of infection, allowing the researchers greater resolution of transcriptional responses compared to whole leaves (Lenzi et al, 2016).…”

Section: Next Generation Of Dual Approachesmentioning

confidence: 99%

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

Naidoo¹,

Visser²,

Zwart³

et al. 2018

Current Issues in Molecular Biology

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RNA-sequencing technology has been widely adopted to investigate host responses during infection with pathogens. Dual RNA-sequencing (RNA-seq) allows the simultaneous capture of pathogen-specific transcripts during infection, providing a more complete view of the interaction. In this review, we focus on the design of dual RNAseq experiments and the application of downstream data analysis to gain biological insight into both sides of the interaction. Recent literature in this area demonstrates the power of the dual RNA-seq approach and shows that it is not limited to model systems where genomic resources are available. Sequencing costs continue to decrease and single cell transcriptomics is becoming more feasible. In combination with proteomics and metabolomics studies, these technological advances are likely to contribute to our understanding of the temporal and spatial aspects of dynamic plant-pathogen interactions. A dual approach in plantaThe interaction between plants and pathogens is an active and dynamic process that can be likened to a duel. Plants have complex defence mechanisms that can be rendered ineffective when pathogens interfere with one of the various processes required for host defence. These processes include penetration resistance, recognition by Pattern Recognition Receptors (PRRs), phytohormone signalling pathways, secretory pathways, secondary metabolite production, and plant cell death (Dou and Zhou, 2012). Until recently, transcriptomic approaches have been applied in the host and pathogen separately to obtain the gene expression profile of each organism and gain insight into infection biology or host defence mechanisms.RNA sequencing (RNA-seq) is a powerful technology that does not rely on any prior knowledge of transcripts and can generate vast quantities of data with much smaller costs involved than for older techniques such as microarrays (Pareek et al., 2011;Wilhelm and Landry, 2009). An advantage of RNA-seq in the field of plant-pathogen interactions is that both plant and pathogen transcripts can be detected simultaneously and accurately in the same sample. This tactic, known as dual RNAseq, in planta RNA-seq, simultaneous RNA-seq, or comparative RNA-seq, is a relatively new technique both in the plant and medical fields. In plants, it allows for the study of plant-pathogen interactions in herbaceous crops Kunjeti et al., 2012;Lowe et al., 2014) as well as trees (Hayden et al., 2014;Liang et al., 2014;Teixeira et al., 2014). This review outlines technical considerations for dual RNA-seq experiments, summarizes recent insights drawn from such approaches in plant-pathogen interactions, and provides an

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Laser microdissection: A promising tool for exploring microorganisms and their interactions with hosts

Cited by 17 publications

References 122 publications

Single-cell level transcriptome of the maize pathogenic fungicochliobolus heterostrophusrace O in infection reveal the virulence related genes, and potential circRNA effector

Single-cell level transcriptome of the maize pathogenic fungicochliobolus heterostrophusrace O in infection reveal the virulence related genes, and potential circRNA effector

Determination of the virulence of single nucleopolyhedrovirus occlusion bodies using a novel laser capture microdissection method

Dual RNA-Sequencing to Elucidate the Plant-Pathogen Duel

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