pFPL Vectors for High-Throughput Protein Localization in Fungi: Detecting Cytoplasmic Accumulation of Putative Effector Proteins

Abstract

As part of a large-scale project whose goal was to identify candidate effector proteins in Magnaporthe oryzae, we developed a suite of vectors that facilitate high-throughput protein localization experiments in fungi. These vectors utilize Gateway recombinational cloning to place a gene's promoter and coding sequences upstream and in frame with enhanced cyan fluorescent protein, green fluorescent protein (GFP), monomeric red fluorescence protein (mRFP), and yellow fluorescent protein or a nucleus-targeted mCHERRY variant. The respective Gateway cassettes were incorporated into Agrobacterium-based plasmids to allow efficient fungal transformation using hygromycin or geneticin resistance selection. mRFP proved to be more sensitive than the GFP spectral variants for monitoring proteins secreted in planta; and extensive testing showed that Gateway-derived fusion proteins produced localization patterns identical to their "directly fused" counterparts. Use of plasmid for fungal protein localization (pFPL) vectors with two different selectable markers provided a convenient way to label fungal cells with different fluorescent proteins. We demonstrate the utility of the pFPL vectors for identifying candidate effector proteins and we highlight a number of important factors that must be taken into consideration when screening for proteins that are translocated across the host plasma membrane.

Gapless genome assembly of Colletotrichum higginsianum reveals chromosome structure and association of transposable elements with secondary metabolite gene clusters

“…The 1.3kb promoter of ChPKS38 was amplified with Phusion polymerase (ThermoFisher Scientific, Waltham, Massachusetts) using primer pair 1 and the 1.2kb 3' flanking region of ChPKS38 with primer pair 2. The mRFP and G418 resistance genes were amplified from the plasmid pFPL-Rg [59] with primer pairs 3 and 4, respectively. The four fragments were then joined using primer pair 5 by double-joint PCR [60].…”

Identification and characterization of suppressors of plant cell death (SPD) effectors from Magnaporthe oryzae

“…Transformation cassettes to observe secretion in rice cells were constructed containing the entire protein coding sequence (including the predicted signal peptide) with its native promoter (1.1 kb) in a translational fusion with mRFP. The genomic sequence of MGG_09379 (SPD8, Bas162) was amplified using primers ISP2_F (aaaaagcaggcttaTTCCGATGATTCCCTCTGTC) and ISP2_R (agaaagctgggtaAGTGCTTTTAACCTGGTCCCA), cloned into the donor vector pDONR, and transferred into the destination vector pFPL‐Rh via Gateway cloning using the described methods (Gong et al ., ). The resulting plasmid (pBV921) was transformed into A. tumefaciens strain AGL1 cells and then incorporated into M. oryzae wild‐type strain O‐137 via Agrobacterium ‐mediated transformation with selection for hygromycin resistance (Khang et al ., ).…”

Interactions between Trichoderma harzianum and defoliating Verticillium dahliae in resistant and susceptible wild olive clones

“…Plasmid pRF‐HUE‐YFP was constructed to express YFP in V. dahliae under the control of the glyceraldehyde 3‐phosphate dehydrogenase constitutive promoter of Aspergillus nidulans . The pair of primers YFP‐UserF (5′‐GGACTTAAUATGGTGAGCAAGGGCGAGGAG‐3′) and YFP‐UserR (5′‐GGGTTTAAUTTACTTGTACAGCTCGTCCATGCCG‐3′) was used to amplify a 720 bp DNA fragment containing the coding region of eYFP using the plasmid pFLG (Gong et al ., ) as template. Both primers included the sequences (underlined letters) needed to ligate the PCR product to plasmid pRF‐HUE digested with Pac I and Nt.Bbv CI USER enzymes (Frandsen et al ., ).…”