Sorghum anthracnose resistance: One MITE to rule them all

Sorghum anthracnose is a highly destructive fungal disease that causes necrotic lesions on leaves and stems. Over time, these lesions can spread to panicles and seeds, resulting in significant yield losses (reviewed in Stutts and Vermerris, 2020). While several anthracnose-resistant sorghum accessions have been identified, these accessions vary widely in their resistance responses as well as in the genomic regions associated with resistance. The specific genes underlying anthracnose resistance and their regulatory mechanisms remain largely unknown. In this issue, Sanghun Lee, Fuyou Fu, and colleagues  Lee et al. (2022) reveal how a miniature inverted-repeat transposable element (MITE) along with other regulators orchestrate the expression of a sorghum immune receptor gene important for anthracnose resistance. Moreover, the authors demonstrate that this gene confers broad-spectrum resistance to at least two other sorghum diseases caused by distinct fungal pathogens, which is quite uncommon for plant immune receptors.

Through screening of sorghum natural variants, Lee et al. (2022) identified SC283, a genotype resistant to the anthracnose disease caused by the fungal pathogen Colletotrichum sublineola. The authors then generated a series of recombinant inbred lines by crossing SC283 with a susceptible genotype (TAM428). Whole-genome resequencing of these recombinant inbred lines revealed a major locus associated with resistance to multiple fungal pathogens: ANTHRACNOSE RESISTANCE GENE 1 (ARG1). ARG1 encodes a protein belonging to the nucleotide-binding leucine-rich repeat (NLR) family of immune receptors. Intriguingly, the ARG1 gene is nested within the intron of a natural antisense transcript: CARRIER of ARG1 (CARG) (see Figure). Transcriptome analysis highlighted several major differences in ARG1 and CARG expression patterns between resistant and susceptible genotypes. Susceptible genotypes express high levels of CARG transcripts while resistant genotypes lack CARG expression. Furthermore, resistant genotypes express high levels of a single full-length ARG1 transcript that encodes all major domains present in a typical NLR protein (coiled-coil domain, nucleotide-binding domain, and leucine-rich repeat domain). However, in susceptible genotypes, a premature stop codon caused by sequence polymorphism in the ARG1 coding region leads to truncated ARG1 proteins that lack the leucine-rich repeat domain. As NLR receptors generally require their leucine-rich repeat domains for pathogen perception and defense activation, loss of this domain likely results in a non-functional ARG1 protein in susceptible genotypes.

Figure.

The ARG1 immune receptor gene associated with sorghum anthracnose resistance is regulated by multilayered and interconnected mechanisms. Unique MITE insertions (indicated by triangles) influence ARG1 and antisense CARG transcript expression. Furthermore, unlike the resistant genotype SC283, the susceptible genotype TAM428 produces a truncated ARG1 protein due to a premature stop codon (indicated with an asterisk) within the ARG1 coding region. TAM428 also produces a smaller splice variant of the ARG1 transcript. Gray and black boxes indicate UTR and coding regions, respectively. Red arrows indicate the positions of the stop codon in the TAM428 ARG1 transcripts. CC, coiled-coil; NB-ARC, nucleotide binding; LRR, leucine-rich repeat domain. Adapted from Lee et al. (2022), Figure 6.

A closer look at the CARG-ARG1 genomic region revealed several distinct MITE-related sequences (see Figure). MITEs are short DNA transposons that tend to insert near genes and can influence gene expression (Feschotte et al., 2002). Lee et al. identified a 275-bp MITE insertion upstream of CARG that is only present in anthracnose-susceptible sorghum genotypes. As resistant genotypes lacking this MITE insertion also do not generate antisense CARG transcripts, it is likely that this 275-bp MITE is necessary for CARG expression. Furthermore, resistant genotypes harbor a unique 420-bp MITE insertion in the ARG1 5′-UTR that may contribute to the high expression of ARG1. The authors tested the effect of the unique 420-bp MITE insertion on reporter gene expression using a series of deletion constructs and confirmed that it acts as a transcriptional activator. Together, these observations suggest that MITEs regulate ARG1 and CARG expression in sorghum genotypes with contrasting anthracnose resistance.

MITEs are rich sources of small RNAs, as they are targeted by the RNA-directed DNA methylation pathway responsible for genome-wide silencing of transposable elements (Feschotte et al., 2002). Although a distinct set of small RNAs was associated with the unique 248-bp MITE present in the 3′-UTR of CARG in susceptible sorghum genotypes, their role in ARG1 regulation remains elusive. Additionally, sense and antisense transcripts can result in double-stranded RNA, leading to the production of small RNAs that can trigger DNA and/or histone methylation (reviewed in Pelechano and Steinmetz, 2013). Exonic regions of CARG were indeed enriched in repressive histone marks (such as H3K9 dimethylation) in resistant genotypes, which is consistent with the low CARG expression pattern observed in these genotypes. An inverse trend was observed for susceptible genotypes with high CARG expression. Based on these findings, it is clear that the sorghum ARG1 gene is regulated by multilayered and interconnected mechanisms. The work by Lee et al. thus provides a strong foundation for further examining the dynamics of ARG1 regulation and function in sorghum resistance against multiple fungal pathogens.