Improved quantification of immune-gold labeling and its use to compare the distribution of cellular factors among sub-chloroplast compartments.

Quantification of immuno-gold labeling can provide valuable information on the quantity and localization of a target within a region of interest (ROI). Background subtraction usually requires preparation of material with a deliberately reduced amount of target component often by gene knockout/knockdown. This paper reports a modified method without the need for gene knockout/knockdown, by using a region outside the ROI as a background and non-immune serum to verify the reliability of the data. An optimized parameter for use in image processing was also developed to improve semi-automatic segmentation of gold particles, by using the standard deviation of pixel intensity together with default parameters (size and intensity) to improve specificity. The modified methods were used to quantify the gold labeling of various components within chloroplasts and their 3 sub-organelle compartments (thylakoid, stroma and starch). Rubisco, actin, myosin, ß-tubulin, Endoplasmic reticulum-retention signal HDEL, Sterol methyltransferase 1, and double stranded RNA were all effectively and consistently quantified at the level of the different sub-chloroplast compartments. The approach should be applicable more widely for high resolution labelling of samples in which a background requiring gene knockout/knockdown is not a realistic option.

Endoplasmic reticulum remodeling induced by Wheat yellow mosaic virus infection studied by transmission electron microscopy.

Plant virus was a kind of organism lived depending on infecting viable host cell and propagated their posterity by replicating its hereditary nucleotide, transcripting into protein, assembling protein and nucleotide into virion (Ortín and Parra, 2006; Sanfaçon, 2005). Viral infection usually induces remodeling of host cell, especially endoplasmic reticulum (ER) for generating membrane packed viral factory. During the infection of Bymovirus, a kind of membranous body (MB) was generated in host cells, which is thought as an ER aggregate. In present study we performed a study on Wheat yellow mosaic virus (WYMV) induced MB by several transmission electron microscopy (TEM) based methods, including cytological observation, component analysis by immuno-gold labeling and structural analysis by electron tomography (ET). WYMV infection induced at least two morphologies of MB, including the lamella dominated morphology (lamella-MB) looked like sprawling cirrus, and the tubule dominated morphology (tubule-MB) looked like latticed network. MB was verified composing of ER as revealed by immuno-gold labeling by antibody against endoplasmic reticulum (ER) retention signal as well as by detailed observation of MB construction modules as double layer membrane. By immuno-gold labeling, both two MB morphologies (lamella-MB and tubule-MB) had same components in viral derived protein and membrane origination (from ER). Structural analysis by ET reconstruction revealed the organization of ER in MB. Lamella-MB was composed of cesER like structures arranged irregularly whereas tubule-MB was composed of tubER like structures arranged regularly. This study provided insights into the structural details in how Bymovirus utilizing host membrane system.