Resistance to Sri Lankan cassava mosaic virus (SLCMV) in genetically engineered cassava cv. KU50 through RNA silencing.

Cassava ranks fifth among the starch producing crops of the world, its annual bioethanol yield is higher than for any other crop. Cassava cultivar KU50, the most widely grown cultivar for non-food purposes is susceptible to Sri Lankan cassava mosaic virus (SLCMV). The objective of this work was to engineer resistance to SLCMV by RNA interference (RNAi) in order to increase biomass yield, an important aspect for bioethanol production. Here, we produced transgenic KU50 lines expressing dsRNA homologous to the region between the AV2 and AV1 of DNA A of SLCMV. High level expression of dsRNA of SLCMV did not induce any growth abnormality in the transgenic plants. Transgenic lines displayed high levels of resistance to SLCMV compared to the wild-type plants and no virus load could be detected in uninoculated new leaves of the infected resistant lines after PCR amplification and RT-PCR analysis. The agronomic performance of the transgenic lines was unimpaired after inoculation with the virus as the plants presented similar growth when compared to the mock inoculated control plants and revealed no apparent reduction in the amount and weight of tubers produced. We show that the resistance is correlated with post-transcriptional gene silencing because of the production of transgene specific siRNA. The results demonstrate that transgenic lines exhibited high levels of resistance to SLCMV. This resistance coupled with the desirable yield components in the transgenic lines makes them better candidates for exploitation in the production of biomass as well as bioethanol

Primers used for amplification.

<p>Primers used for amplification.</p

Sri Lankan cassava mosaic virus (SLCMV) symptoms severity score by visual detection in wild-type and transgenic plants.

<p>^aThree plants were tested per transgenic line.</p><p>^bData represent the sum for the three plants tested and were recorded at 45 days post inoculation.</p><p>^cDisease indices were calculated using the equation shown in materials and methods.</p><p>Disease rating is based on the calculated disease indices. Symptom severity score, the intensity of the disease on the leaf is expressed on a scale of 0–3 grades (0, no symptom; 1, mild/faint mosaic symptoms; 2, moderate mosaic symptoms; 3, severe mosaic symptoms, as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120551#pone.0120551.g007" target="_blank">Fig. 7</a>). Plants with a disease index of 0% were considered as immune, those with a disease index <25% as having high levels of resistance (HR), those with a disease index of 25.1–50.0% as being moderately resistant (MR), those with a disease index of 50.1–75.0% as susceptible (S), and those with a disease index of 75.1–100% as highly susceptible (HS), under the period of study. <i>NA</i> not applicable, <i>WT</i> wild-type plant, <i>MC</i> mock inoculated control plant, <i>K3-K10</i> transgenic cassava lines expressing hairpin SLCMV.</p><p>Sri Lankan cassava mosaic virus (SLCMV) symptoms severity score by visual detection in wild-type and transgenic plants.</p

Plant height and yield characteristics in Sri Lankan cassava mosaic virus (SLCMV) infected transgenic and wild-type plants 60 days post inoculation.

<p>Means with the same case letter in a given vertical array indicate no significant difference at 5% probability level using least significant difference (LSD) test. <i>WT</i> wild-type plant, <i>MC</i> mock inoculated control plant, <i>K3-K10</i> transgenic cassava lines expressing hairpin SLCMV. <i>NA</i> not applicable</p><p>Plant height and yield characteristics in Sri Lankan cassava mosaic virus (SLCMV) infected transgenic and wild-type plants 60 days post inoculation.</p

Cassava tubers harvested form plants inoculated with SLCMV.

<p>The tubers were harvested 60 days after inoculation. WT, non-transformed wild-type plant. K4 and K7 transgenic cassava lines expressing hairpin SLCMV AV2::AV1. MC, mock inoculated control plant.</p

Detection of SLCMV specific siRNA in cassava plants before (A) and after (B) SLCMV inoculation.

<p>The inoculated plants were analyzed for siRNA accumulation at 60 days post inoculation (dpi). The lower panel shows the loading level of each sample after ethidium bromide staining. WT, Inoculated wild-type plant. K3-K10, transgenic lines expressing hairpin SLCMV AV2::AV1.</p

Detection of 527 bp <i>SLCMV</i> and 700 bp <i>nptII</i> genes in transgenic cassava lines.

<p>Lane M, molecular size marker (ø174HaeIII digests). Lane P, positive control (plasmid DNA). Lane WT, non-transformed wild-type control plant. Lanes K3–K10, independent transgenic cassava lines.</p

Screening of transgenic plants for detection of viral load in plants inoculated with SLCMV.

<p>PCR amplification (A) and RT-PCR analysis (B) of a fragment of SLCMV (DNA-A) in emerging new leaves of transgenic lines 60 days after infection with the virus. The lower panel represents rice actin (RAc1) gene used as an internal control for RNA input for RT-PCR.</p

Screening of transgenic cassava lines for resistance to Sri Lankan cassava mosaic virus.

<p>Four week-old hardened transgenic and wild-type plants lines were mechanically inoculated twice with SLCMV. Symptom development was assessed every alternate day and photographs were taken at 7, 14 and 28 days post inoculation (dpi). WT, non-transformed wild-type plant. K3, susceptible transgenic line. K4 resistant transgenic line. Arrow indicates leaf deformation.</p

Main stages of SE production and plant regeneration in cassava cultivar KU50.

<p>Main stages of SE production and plant regeneration in cassava cultivar KU50.</p