High throughput virus-induced gene silencing implicates heat shock protein 90 in plant disease resistance

Abstract

Virus‐induced gene silencing was used to assess the function of random Nicotiana benthamiana cDNAs in disease resistance. Out of 4992 cDNAs tested from a normalized library, there were 79 that suppressed a hypersensitive response (HR) associated with Pto‐mediated resistance against Pseudomonas syringae. However, only six of these clones blocked the Pto‐mediated suppression of P.syringae growth. The three clones giving the strongest loss of Pto resistance had inserts corresponding to HSP90 and also caused loss of Rx‐mediated resistance against potato virus X and N‐mediated tobacco mosaic virus resistance. The role of HSP90 as a cofactor of disease resistance is associated with stabilization of Rx protein levels and could be accounted for in part by SGT1 and other cofactors of disease resistance acting as co‐chaperones. This approach illustrates the potential benefits and limitations of RNA silencing in forward screens of gene function in plants.

Plant Immunity: The Origami of Receptor Activation

“…So far, genetic approaches have identified relatively few components required for R gene function in plants [5]. A highly conserved protein class, the heat-shock proteins, has now been added to the list and work on these proteins promises to provide fresh insights to the molecular mechanics of the pathogen recognition process [6][7][8][9].…”

“…An indication that the former might be true is the observation that RPM1 steady-state levels are greatly decreased or undetectable in non-challenged rar1 or hsp90.2 mutant backgrounds [7,19]. The role of SGT1 in R protein function is likely to be different, because RX protein accumulation was impaired following NbHSP90 silencing but unaffected upon NbSGT1 silencing [9].…”

Differential Requirement of Oryza sativa RAR1 in Immune Receptor-Mediated Resistance of Rice to Magnaporthe oryzae

“…The function of these proteins in disease resistance responses have been extensively investigated previously using mutant analyses Chandra-Shekara et al, 2004;Hubert el al., 2003;Lu et al, 2003;Shirasu et al, 1999;Takahashi et al, 2003) and by virus induced gene silencing (VIGS) analyses in several plant species (Bhattarai et al, 2007;de la Fuente van Bentem et al, 2005;Leister et al, 2005;Liu et al, 2004;Scofield et al, 2005). The data from these studies revealed differing specificities for these proteins in diverse NB-LRR-mediated resistance responses.…”

“…For example, RAR1 and SGT1 are required for MLA6-and MLA12-, but not MLA1-, mediated resistance (Azevedo et al, 2002). Tobacco N, a Tobacco mosaic virus resistance protein, and wheat Lr21, a Puccinia triticina resistance protein, require each of the proteins for the initiation of immune responses against pathogens (Liu et al, 2004;Lu et al, 2003;Peart et al, 2002;Scofield et al, 2005). The function of each of these chaperone proteins has not yet been fully explored in rice immune responses governed by NB-LRRs.…”

Transient Virus Expression Systems for Recombinant Protein Expression in Dicot- and Monocotyledonous Plants

“…In this approach, cDNA libraries are constructed within virus expression vectors and gene function to be assessed by screening infected plant hosts for phenotypic or metabolic changes measured by various input and output focused screening assays. TMV and PVX VIGS vectors were the first to employed in this manner in the dicot N. benthamiana, with BSMV used shortly after in the monocot barley [84,100]. Since these first reports, vector systems for functional genomics have continued to be refined and optimized in an expanding number of hosts [18,87], including systems amenable for high throughput screens in monocots based on BSMV [101,102].…”