RNA silencing approaches for identifying pathogenicity and virulence elements towards engineering crop resistance to plant pathogenic fungi.

Abstract

Over the past several years, RNA interference (RNAi) has emerged as a powerful genetic tool that is being exploited not only in fundamental research for the assessment of gene functions, but also in various fields of applied research in medicine and agriculture. In plants, RNAi strategies have the potential to allow manipulation of various aspects of food quality and nutritional content, but also for plant protection. In the study of plant-fungus interactions, a comprehensive understanding of pathogenic mechanisms based on information from genome sequences demands advanced genomics tools and infrastructure. In order to make use of the plethora of information yielded by the rapidly increasing fungal pathogen genome sequencing studies, there is a growing need to achieve gene function assessment amenable to high-throughput techniques. Targeted gene disruption is effective but may not be feasible in certain fungi because of lack of transformation protocols or their life styles. RNAi techniques offer an alternative but, in contrast to other biological systems, only few studies have reported the use of RNAi to explore gene function in plant pathogenic fungi. In this review, we summarize a variety of options using RNAi techniques that have become available to study gene function for high-throughput phenotypic screening of virulence and pathogenicity factors in plant fungi. The identification of such genes can be exploited for the protection of crops against fungal pathogens.

Host‐induced silencing of essential genes in Puccinia triticina through transgenic expression of RNAi sequences reduces severity of leaf rust infection in wheat

“…This technique, termed post‐transcriptional gene silencing (PTGS) in plants, involves the endonucleolytic cleavage of double‐stranded RNA (dsRNA) into small, ~20–23 nucleotide (nt)‐long, small interfering RNA (siRNA) duplexes by the Dicer‐like (DCL) enzymes which along with their associated proteins control the expression of genetic information (Baulcombe, ; Wilson and Doudna, ). An RNAi‐based approach referred to as host‐induced gene silencing (HIGS) or host‐delivered RNAi (HD‐RNAi) in which small RNAs are produced by the host plant to target parasite transcripts has provided a promising strategy for improving plant resistance against pathogens, including fungi and oomycetes (Andrade et al ., ; Govindarajulu et al ., ; Huang et al ., ; Jahan et al ., ; Koch and Kogel, ; Koch et al ., ; Nowara et al ., ; Panwar et al ., , ; Song and Thomma, ; Yin et al ., ). When targeted against crucial pathogenicity genes, HD‐RNAi can potentially be developed as a genetic method to curb pathogen virulence for pesticide‐free disease control in crop plants.…”

RNAi as an emerging approach to control Fusarium head blight disease and mycotoxin contamination in cereals

“…24 This approach utilised a RNAi vector containing an intron sequence between two inversely oriented and self-complementary target sequences, which when expressed, generate a dsRNA molecule with a hairpin structure. 26 In plants, and some animals, locally initiated gene silencing can spread to other parts of the organisms, through systemic or cell-to-cell transport of the silencing signal. In plants, the silencing signal is suggested to be transmitted long range by the phloem, following source to sink dynamics.…”

“…By carefully designing the sequences to be used for HIGS and targeting the same gene in different fungal species, there is a strong possibility that broad spectrum control could be achieved. 26 Exploring the vast data sets of genomic and transcriptomic information during the initial construct design phase in any project should decrease the chances of off-target silencing of unintended genes in the host plants, as well as in the beneficial plant-associated organisms, such as mycorrhizas, rhizobia and biocontrol species, like Trichoderma species. As a example, a HIGS study conducted in maize to downregulate aflatoxin biosynthesis in Aspergillus flavus caused stunting and reduced kernel placement in transgenic plants, potentially due to off-target silencing of other genes.…”

“…Multiple 'essential for life' genes have already been identified and published for plant pathogenic species and these could be the first targets for RNAi. 26 Not all fungal species may be targeted through HIGS. Some fungi species apparently lack the whole or most of the RNA silencing components in the genome.…”

“…floral spikes and not leaves or roots). 26 HIGS approaches are unlikely to function post harvest to combat infections occurring in dried seeds, leaves, fruits and/or root. This is because of low overall plant physiological and metabolic activities and therefore limited opportunities to initiate and then systemically propagate the underlying silencing mechanisms.…”