The generation of self-complementary RNA, whether through antisense RNA transcription or hairpin formation, triggers a sequence-specific mRNA degradation, leading to gene silencing. RNAi signals are diffusible and can traverse across source-to-sink gradients (Brosnan and Voinnet, 2011). In plants, the transgene silencing signals mainly move short distances from cell to cell, but can also spread over long distances through the vasculature, the latter process being mostly evident through grafting experiments (Palauqui et al., 1997; Dunoyer et al., 2010a). Like other eukaryotes, most fungi are also sensitive to RNAi and recently it was shown that silencing signals can travel from host cell to fungus, presumably through the haustorial interface (Nowara et al., 2010; Yin et al., 2011). Uptake of genetic material, in the form of RNA, has also been demonstrated between host plants and parasites such as insects, nematodes or other parasitic plants feeding on them (Huang et al., 2006; Baum et al., 2007; Mao et al., 2007; Tomilov et al., 2008). However, the mechanism behind such cross-species transfer of silencing molecules remains to be elucidated. Our data show that in the Puccinia-wheat pathosystems, the silencing signals are likely to be the siRNA molecules, as these were generated in the host cells (Figures 2 and 3). Preliminary studies suggest that translocation of genetic material from host to infecting fungal cells may occur via the exosomal biogenesis pathway (Casadevall et al., 2009; Meyer et al., 2009; Lu et al., 2012). At sites of fungal penetration, multivesicular compartments, including multivesicular bodies (MVBs) and cell wall-associated paramural bodies, aggregate in host cytoplasm around haustorial complexes, facilitating an anterograde, and probably retrograde, polarized vesicle trafficking across the plant–pathogen cellular interface. These MVBs contain multiple intraluminal vesicles (ILVs), which upon fusion with the plasma membrane are released extracellularly as exosomes into the paramural space (Meyer et al., 2009; Lu et al., 2012). Interestingly, exosomes have been shown to contain both mRNA and non-coding small RNAs that can be delivered, and can be functional, in recipient cells, thereby facilitating genetic exchange between cells (Valadi et al., 2007). Transport of RNA by exosomes might require appropriate receptors at the cell surface for attachment, or follow other specialized transfer pathways. MVB-like compartments have been reported in trafficking mechanisms at intercellular channels called gap junctions, nanotubes or even internalization of sections of plasma membrane by neighbouring cells (Gibbings and Voinnet, 2010). It is possible that in our system the siRNA species generated in the host ‘silencing donor’ are transferred into the ‘fungal recipient’ through this exocytic/endocytic exchange mechanism at the haustorium interface (Figure 7). During plant infection, biotrophic and hemibiotrophic fungi secrete effector proteins, some of which translocate to the plant apoplast or cytosol where they can alter host responses to condition susceptibility. Recently, it was reported that some effectors secreted by oomycete pathogens, which have infection strategies similar to rust fungi, may enter host cells via receptor-mediated endocytosis (Kale and Tyler, 2011). However, similar effector delivery has not yet been discovered for biotrophic fungi. It is currently unknown whether a bidirectional trafficking across the host–parasite cellular interface can take place, and whether this could include small RNA species. Nutrient uptake has been reported to occur in bean rust fungal haustoria through specific transporters (Voegele and Mendgen, 2003), and it is possible that siRNAs use these or similar transporters. Alternatively, their sizes may be sufficiently small as to allow other (passive) ways for crossing various membranes and the EHM.
Figure 7. Schematic presentation of possible in planta-induced transient gene silencing (PITGS) mechanisms by which siRNA molecules generated in host cells could be delivered into fungal cells. Fungal dsRNA, produced inside host cells, is cleaved by the plant silencing machinery using endonuclease-type DICER enzymes into small silencing molecules (siRNAs). Plasma membrane proteins (PMPs) and other specific materials destined for extracellular secretion are internalized by endocytosis, trapping small RNAs along the way to form sorting endosomes that subsequently get compartmentalized as intraluminal vesicles (ILVs) within multivascular bodies (MVBs). These MVBs can either fuse with lysosomes to permit degradation of the ILVs or follow a retrograde pathway, fusing with the plasma membrane (PM) and releasing the ILVs as exsosomes into the paramural space. This space acts as a nexus of communication and transport between host or pathogen cells and their respective exterior environments. Passage through the haustorial cell wall (HCW), either active or passive, occurs and once inside the fungal haustorium the silencing molecules trigger RNAi of their mRNA targets, and may act as primers in the fungal silencing pathway, leading to the generation of systemic silencing signals. HCW, haustorial cell wall; PCW, plant cell wall; HMC, haustorium mother cell; SSV, substomatal vesicle; UD, urediniospore.
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Genomic resources for the agronomically important cereal rust fungi are being generated at a fast pace, yet functional assay systems are lacking to test for genes predicted to be involved in pathogenicity or virulence in these obligate biotrophic pathogens. We made use of an RNAi approach by developing a functional A. tumefaciens-mediated PITGS assay for wheat that should allow the rapid testing of many genes. Using this approach we demonstrated the silencing of three endogenous Pt genes, suggesting the feasibility of this system to study loss-of-function phenotypes in rust fungi. Gene silencing by transient expression of hpRNA does not require stable genetic transformation, and is consequently a promising technique for functional genomic studies in organisms refractory to genetic manipulation. Our results demonstrate that wheat-expressed fungal double-stranded RNA (dsRNA) can trigger RNAi of corresponding homologous genes in several Puccinia species. Furthermore, silencing of PtMAPK1, PtCYC1 and PtCNB results in significant disease suppression of three major wheat rust pathogens, showing that these genes and the signaling cascades they control might be important in the disease process, and suggesting that they could be excellent targets to generate durable and broad-spectrum genetic resistance against these destructive fungi. Pyramiding multiple gene targets for silencing could provide further disease suppression, and could potentially be used for rust disease control in wheat.