RXLR-Mediated Entry of Phytophthora sojae Effector Avr1b into Soybean Cells Does Not Require Pathogen-Encoded Machinery

Abstract

Effector proteins secreted by oomycete and fungal pathogens have been inferred to enter host cells, where they interact with host resistance gene products. Using the effector protein Avr1b of Phytophthora sojae, an oomycete pathogen of soybean (Glycine max), we show that a pair of sequence motifs, RXLR and dEER, plus surrounding sequences, are both necessary and sufficient to deliver the protein into plant cells. Particle bombardment experiments demonstrate that these motifs function in the absence of the pathogen, indicating that no additional pathogen-encoded machinery is required for effector protein entry into host cells. Furthermore, fusion of the Avr1b RXLR-dEER domain to green fluorescent protein (GFP) allows GFP to enter soybean root cells autonomously. The conclusion that RXLR and dEER serve to transduce oomycete effectors into host cells indicates that the >370 RXLR-dEER–containing proteins encoded in the genome sequence of P. sojae are candidate effectors. We further show that the RXLR and dEER motifs can be replaced by the closely related erythrocyte targeting signals found in effector proteins of Plasmodium, the protozoan that causes malaria in humans. Mutational analysis of the RXLR motif shows that the required residues are very similar in the motifs of Plasmodium and Phytophthora. Thus, the machinery of the hosts (soybean and human) targeted by the effectors may be very ancient.

Microbe-Independent Entry of Oomycete RxLR Effectors and Fungal RxLR-Like Effectors Into Plant and Animal Cells Is Specific and Reproducible

“…Plasmolysis of the root cells confirmed that the protein was inside the cells. confirmed the root cell entry experiments of Dou et al (2008) using full length Avr1b protein and using more specific mutations of RXLR2 (qFLR) that agreed with results of bombardment experiments of Dou et al (2008). Furthermore, validating an observation of Shan et al (2004), showed that full length Avr1b protein could trigger cell death on leaves containing the Rps1b resistance gene, but not on leaves lacking Rps1b.…”

“…The authors, from the van West, Kahmann and Nuernberger labs, presented data in support of the conclusion that the RxLR domains of P. infestans Avr3a and of P. sojae Avr1b alone are NOT sufficient to enable microbe-independent entry of proteins into host and non-host plant and animal cells. More specifically, they reported that they were unsuccessful in their attempts to reproduce key experiments previously reported by Dou et al (2008) and in which GFP fusions to Avr1b and its RxLR domain were observed to enter soybean root cells and human lung epithelial cells. Instead, they reported that any fluorescent protein can be observed to enter soybean roots if exposed to the roots for an extended period.…”

“…The first are plant transient expression experiments, by bombardment or agroinfiltration, in which the effector in question is secreted from the plant and then must re-enter to trigger a measured response (usually cell death in the presence of a cognate R gene) (Dou et al, 2008;Rafiqi et al, 2010;Gu et al, 2011;Anderson et al, 2012;Sun et al, 2013). The second is to expose root tips or leaf tissue to purified effectors and then, to measure entry microscopically using antibodies, or by the presence of a chemical or protein fluorescent label attached to the effector (Dou et al, 2008;Plett et al, 2011;Wawra et al, 2012). In the case of P. sojae effector Avr1b, the same RxLR and dEER motif mutations that abolished entry by the effectors from the P. sojae transformants also abolished entry as measured by the transient expression and purified protein assays (Dou et al, 2008;, supporting the conclusion that these assays are relevant to infection in vivo.…”

“…The second is to expose root tips or leaf tissue to purified effectors and then, to measure entry microscopically using antibodies, or by the presence of a chemical or protein fluorescent label attached to the effector (Dou et al, 2008;Plett et al, 2011;Wawra et al, 2012). In the case of P. sojae effector Avr1b, the same RxLR and dEER motif mutations that abolished entry by the effectors from the P. sojae transformants also abolished entry as measured by the transient expression and purified protein assays (Dou et al, 2008;, supporting the conclusion that these assays are relevant to infection in vivo. Similar experiments have also identified fungal effectors that appear to enter plant cells in the absence of the microbe, via RxLR-like motifs ([R, K, H]×[L, M, I, F, W, Y]); with no dEER-like motifs) Gu et al, 2011;Plett et al, 2011).…”

Predicted Effector Molecules in the Salivary Secretome of the Pea Aphid (Acyrthosiphon pisum): A Dual Transcriptomic/Proteomic Approach

“…Many pathogens have specific secretion systems that enable the delivery of these effectors, such as the type III secretion system in bacteria [15][16][17] or the RXLR system in oomycetes. [18][19][20] By the nature of their feeding, aphids are thought to deliver effectors in their saliva, [21][22][23] as is the case for plant pathogenic nematodes. [24][25][26] However, little is known about the bioactive components of aphid saliva.…”