Studies on Resistance Mechanisms in Cassava Genotypes to Geminiviruses

Abstract

Through classical genetics three sources of CMD resistance, polygenic recessive resistance designated CMD1, the dominant monogenic type named CMD2 and the newly identified CMD3 are being introgressed in cassava genotypes to combat CMD. However, the genes and mechanisms involved in inherent resistance to CMD remain unknown. To unravel the mode of innate CMD resistant, cassava genotypes carrying CMD1 (TMS 30572), CMD2 (TME 3, TME 7, TME 14 and TME 204) and CMD3 (TMS 97/2205 and TMS 98/0505) resistant loci and CMD susceptible genotypes (60444 and Ebwanatereka) were studied. In an effort to identify sequences of genes harbored in genomic region carrying CMD2 locus, the existing TME 3 (CMD2 type) bacterial artificial chromosomes (BAC) libraries were hybridized with each of the marker probe on a high density colony filters. A total of 130 BAC clones were identified with CMD2 flanking markers. Among them 23 clones were positive for at least two markers. Through whole BAC sequencing, contigs of up to 100kb were assembled and anchored on markers flanking CMD2 locus on either side of the locus. Using BAC sequencing five disease resistance genes were identified and confirmed in different cassava genotypes although they were mapped outside of CMD2 locus region. Cassava genotypes responded differently to biolistic infection with African cassava mosaic virus (ACMV-CM) and East African cassava mosaic virus (EACMV KE2 [K201]). Cassava genotypes carrying innate resistant showed high levels of resistance to ACMV-CM, with viral DNA undetectable by PCR beyond 7 days post inoculation (dpi). Contrastingly, all cassava genotypes developed severe symptoms after systemic infection with EACMV KE2 (K201) corresponding with high viral titer. However, complete recovery from EACMV KE2 (K201) infection was observed in CMD1, CMD2 and CMD3 genotypes by 65 dpi with no detectable virus in newly formed leaves. Overall the highest level of CMD resistant was observed in cassava landrace TMS 97/2205 carrying CMD3 resistance locus. Virus derived small RNAs (vsRNAs) of 21 – 24 nt length and distributed throughout the entire virus genome in sense and antisense polarities were identified through small RNAs deep sequencing. The proportion of vsRNAs reads was; 21nt (45%), 22 nt (28%) and 24 nt (18%) in all genotypes studied and were directly correlated with virus titer and CMD symptoms. Variation in abundance of 5’ nt among different vsRNAs populations indicated involvement of multiple argonaute (AGO) protein complexes in antiviral defense. Virus induced gene silencing (VIGs) of cassava homologs of CHROMOMETHYLTRANSFERASE 3 (CMT3) (ManesCMT3) and METHYLTRANSFERASE 1 (MET1) (ManesMET1) intensified EACMV pathogenicity and abolished classical recovery in CMD1 and CMD2 cassava genotypes indicating the role of CHG and CG methylation in antiviral defense. The efficacy of VIGs in suppression CMT3 and MET1 was higher in CMD susceptible cassava genotypes whereby >85% knock down was achieved compared with 50% down regulation in CMD resistant cassava genotypes. Pleiotropic phenotype was induced by knock down of ManesCMT3 and ManesMET1 in all cassava genotypes. Filiform leaves, a phenocopy of severe CMD infection was induced by knock down of ManesCMT3. Significantly (P≥0.05) higher virus titer was observed for cassava genotypes TMS 30572, TME 14, Ebwanatereka and 60444 indicating the importance of establishment methylation in geminiviruses defense.