植物生理

Gene expression profiling studies are commonly used to study signaling pathways and their impact on transcriptional regulation in plants. In some cases, a profiling study results in expression profiles in which most genes exhibit a small number of differentially expressed states amo ...

Ubiquitination is one of the most important posttranslational modifications in all eukaryote organisms. Ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligase (E3) are the three key enzymes in this process. To detect the specificity between ...

The ubiquitination/proteasome system is involved in nearly all plant signaling processes. Many signaling components are degraded by the 26S proteasome upon ubiquitination by specific E3 ubiquitin ligase. However, due to technical limitations, only a few pairs of E3 ligase–substr ...

Proteins generally interact with some other proteins to achieve their cellular functions. Fluorescence resonance energy transfer (FRET) microscopy provides a powerful technique to elucidate such interactions in vivo. FRET occurs when two properly chosen fluorophores are su ...

One of the primary roles of the cell surface is to provide an effective barrier to various external environmental factors. Specifically, the surface properties of organisms serve as a critical obstacle to pathogen attack. Since its inception, Atomic Force Microscopy (AFM) has enabled nan ...

Rho-family small GTPases are conserved molecular switches of signaling networks in eukaryotic cells, and regulate many cellular responses, such as cytoskeletal reorganization, gene expression, and polarized vesicular trafficking. To understand the functions of Rho GTPase, ...

The yeast two-hybrid (Y2H) system is a binary method widely used to determine direct interactions between paired proteins. Although having certain limitations, this method has become one of the two main systemic tools (along with affinity purification/mass spectrometry) for intera ...

Recently, great strides have been made in the area of host-pathogen interactions involving necrotrophic fungi. In this article we describe a method to identify, produce, and characterize effectors that are important in host–necrotrophic fungal pathogen interactions, and to genet ...

In this chapter a method for the heterologous production of fungal proteins in the yeast Pichia pastoris is described. Starting with cloning of the sequence encoding the gene of interest into the expression vector, this protocol describes P. pastoris transformation, production of the pro ...

Laser microdissection (LM) of plant tissues infected with a fluorescent protein-tagged fungus is a useful method for obtaining samples highly enriched in fungal RNA for downstream analysis such as hybridization to a microarray. This paper outlines the requirements for successful LM ...

Proteomics and transcriptomics are established functional genomics tools commonly used to study filamentous fungi. Metabolomics has recently emerged as another option to complement existing techniques and provide detailed information on metabolic regulation and seco ...

Next-Generation Sequencing (NGS) methods have revolutionized various aspects of genomics including transcriptome analysis. Digital expression analysis is all set to replace analog expression analysis that uses microarray chips through their cost-effectiveness, rep ...

Targeted gene replacement is one of the primary strategies for functional characterization of fungal genes and several methods have been developed for this purpose over the years. The increased availability of genome sequence information in the present times has enabled wider adopt ...

Microarrays are a valuable technology to study fungal physiology on a transcriptomic level. Various microarray platforms are available comprising both single and two channel arrays. Despite different technologies, preprocessing of microarray data generally includes qua ...

The development of confocal microscopy and its application to studies of plant–pathogen interactions have revolutionised research into the role of selected molecules and cell components in pathogen infection strategies and plant defence responses. Confocal microscopy all ...

Accurate evaluation of disease phenotypes is considered a key step to study plant–microbe interactions, as the rate of host colonization by the pathogenic microbe directly reflects whether the defense response of the plant is compromised. Although several techniques were develop ...

RNA-mediated gene silencing is one of the major tools for functional genomics in fungi and can be achieved by transformation with constructs that express hairpin (hp) RNA with sequences homologous to the target gene(s). To make an hpRNA expression construct, a portion of the target gene can be ampl ...

Activity-based protein profiling (ABPP) is a powerful analytical method to detect and compare the activity of proteins in proteomes. This is achieved using specific activity-based probes that are often derived from inhibitors and are linked to reporter groups like rhodamine or biotin f ...

Gene cloning is the first step of targeted gene replacement for functional studies, discovery of gene alleles, and gene expression among other applications. In this chapter, we will describe a cloning technique suitable for fungal species where the genomic information and sequences ava ...

Upon infection of plants by pathogens, at least at the early stages of infection, the interaction between the two organisms occurs in the apoplast. To study the molecular basis of host susceptibility vs. resistance on the one hand, and pathogen virulence vs. avirulence on the other, the identific ...