Background
The starchy storage root of cassava serves as a staple food for millions of people in Africa. In 2014, over 50% of the world’s cassava production took place in sub-Saharan Africa, where 146.8 million tons were harvested [
1]. While cassava is resilient to abiotic stresses such as prolonged drought [
2], its production is constrained by the two viral diseases, cassava mosaic disease (CMD) and cassava brown streak disease (CBSD) [
3]. Cassava mosaic disease is caused by a cassava mosaic geminivirus (CMG) complex. CMGs are single-stranded bipartite DNA viruses in the family Geminiviridae, genus
Begomovirus, comprised of 11 species, two of which are present in the Indian sub-continent, with the rest endemic to Africa [
4,
5].
Improvement programs for development of CMD-resistant cassava germplasm include introgression of polygenic recessive resistance from the related species
Manihot glaziovii (CMD1), identification of monogenic dominant resistance in West African cassava landraces (CMD2), and more recently, production of highly resistant cultivars carrying a quantitative trait loci (CMD3) [
6‐
9]. Screening cassava germplasm for resistance to CMD traditionally involves cultivation under field conditions with exposure to transmission of CMGs mediated by the whitefly vector
Bemisia tabaci for a growth cycle of 12–18 months [
6,
10,
11]. Under contained conditions in the greenhouse or growth chamber, inoculation of cassava with CMGs can be achieved by a) graft inoculation from a CMG-infected host to healthy plants [
12‐
14]; b) delivery of DNA genomes as infectious clones via microparticle bombardment [
15‐
17]; c) A
grobacterium-mediated inoculation of plants with cloned infectious DNA genomes [
18]; or d) mechanical transmission of cloned viral DNA genomes by abrasion [
19]. Irrespective of the inoculation method employed, CMD symptom development and severity is scored over a period lasting 12–22 weeks from the time of inoculation through the potential disease recovery process [
15,
18]. During this time the resistant/tolerant cultivars are identified based on displayed recovery phenotype on newly formed leaves, while the susceptible cultivars remain symptomatic throughout.
Methods currently available for evaluating resistance and susceptibility to CMD in new cultivars, breeding lines or transgenic and gene edited events are therefore lengthy, space inefficient and require frequent assessment of leaf symptoms by skilled personnel. We report here the development of a simple screening system for determining resistance or susceptibility to CMD that can be completed within 2–4 weeks from the time of inoculation. This rapid screening system is based on virus-induced gene silencing (VIGS) of an endogenous MeSPY gene. The method described saves time and space in the greenhouse and enhances capability to allow screening of a large number of plants in a short period of time.
Discussion
Virus-Induced Gene Silencing (VIGS) has been used both in model and non-model plant systems to elucidate gene function [
16,
32‐
35]. In cassava, a VIGS system was first reported based on an isolate of
African cassava mosaic Cameroon virus (ACMV-CM) [
16]. It was reported previously that this ACMV-CM infectious clone is less virulent than EACMV-K201 such that the CMD2-type cultivars TME 204, TME 3 and TME 7 (Oko-iyawo) are infected at low frequencies (0–30%) and develop only mild disease symptoms [
15,
28]. This is less than ideal if robust suppression of gene expression is desired within an experimental system. The
East African cassava mosaic virus isolate EACMV-K201 [
21] is highly virulent and has been shown to infect all cassava genotypes [
15,
28]. A new VIGS system was developed, therefore, based on EACMV-K201 by cloning target sequences into the coding region of the CP gene of the DNA-A component. Efficacy of this VIGS vector was confirmed by silencing the endogenous
MePSY2 gene in the CMD-susceptible cultivar TME 7S, resulting in visually detectable bleaching and production of chlorotic tissues throughout a 12-week observation period (Fig.
1b).
Screening cassava germplasm for resistance to CMD under field conditions requires many months [
10,
36]. Under laboratory or greenhouse conditions this evaluation period is shorter but still needs 12–22 weeks [
15,
18] to allow for full disease establishment and expression of the recovery phenotype typical for most cassava cultivars. Data presented here shows that the MeSPY1-VIGS system can be used as a quick screening tool to determine resistance and susceptibility to CMD (Figs.
2,
3 and
4). This was achieved by targeting the cassava
SPY gene using a newly developed EACMV-K201-based VIGS vector delivered by biolistic inoculation. The established CMD scoring system for cassava involves visual assessment of symptoms based on a scale of 0–5 [
25]. Experienced personnel are required to capture accurate data due to subtle presentation of disease symptoms in some cultivars. The symptom scores are recorded for each individual plant in an experiment often at a frequency of 1–2 times a week for 12–22 weeks depending on the cultivar, virus species and isolate used. Using the MeSPY1-VIGS screening system, only shoot-tip necrosis/death or whole plant death needs be scored, and only once or twice up to 4 weeks post inoculation. On average, this saves 8–18 weeks per inoculation experiment allowing 3–5 times more plants to be tested in the same time period and the same available greenhouse space. This MeSPY-VIGS screening system has been developed using known CMD-resistant and CMD-susceptible cassava cultivars and plant lines that have been well characterized under field and greenhouse conditions [
8,
11,
15,
28]. Data generated (Figs.
1,
2,
3 and
4) corroborates accurately with the known CMD response of these cultivars, as further validated with challenge experiment using GFP-VIGS (Fig.
5), showing the robustness of the screening system.
The cause of shoot-tip necrosis that eventually leads to whole plant death in CMD-susceptible cassava lines is not clear. Both virus DNA and
MeSPY transcript quantification showed significant differences between the CMD-resistant and CMD-susceptible plant lines, with CMD-susceptible plant lines having greater virus load than resistant lines (Fig.
6a and
b) in a manner consistent with our previous report [
15]. This viral load corresponded with an expected and significant reduction in
MeSPY transcript in CMD-susceptible plants (Fig.
6c).
SPY is involved in diverse physiological and developmental roles, including suppression of GA signaling [
30], promotion of cytokinin response [
37,
38] and enhancement of sensitivity to drought and salinity stress [
39]. A
SPY mutation in Arabidopsis caused elongation of the stem that phenocopies wild-type plants treated with GA [
29,
30]. In Arabidopsis,
SPY and
SECRET AGENT (SEC) [
40,
41] are the only proteins known to have
O-linked N-acetylglucosamine transferase activity involved in posttranslational modification of other proteins. We tested if silencing of cassava
SEC (
MeSEC) mimics what has been observed in plants challenged with MeSPY1-VIGS as shown in this study (Figs.
1,
2,
3 and
4). Silencing of the cassava ortholog of Arabidopsis
SEC did not cause lethality or any different phenotype in susceptible or resistant TME 204 and TME 7 cassava cultivars as compared to GFP-VIGS (data not shown), suggesting that
MeSPY and
MeSEC may not be functionally redundant. The cause of death after challenge with MeSPY1-VIGSs might therefore be due to the crucial role
MeSPY plays in plant function and/or due to an unknown interaction with the geminiviruses. Transgenic plants overexpressing
MeSPY and RNAi cassava lines are being recovered to further elucidate a putative role of
SPY in CMD resistance.
The ability to discriminate between CMD-resistant and-susceptible lines using a simple MeSPY1-VIGS challenge has significant practical application. We recently reported that the CMD2-type cultivars TME3, TME 7 and TME 204 lose inherent resistance to CMD after passage through the process of somatic embryogenesis, and that this phenomenon occurs at an early stage after culture of an explant on auxin-containing media [
31]. Meristem tip culture for virus elimination in infected cassava plants to allow movement of plants between countries or within a country, in vitro germplasm preservation and production of transgenic and gene edited plants often employ tissue culture procedures [
42,
43]. The rapid CMD screening system developed in this study can easily be applied for determining preservation of CMD resistance after such tissue culture manipulations. Furthermore, the technique can be applied for high throughput screening of large numbers of progenies in a breeding pipeline under controlled greenhouse conditions. Besides understanding the molecular mechanisms involved in the phenotype observed in susceptible cassava lines reported here, investigation into the utility of SPY-VIGS as a screening tool in other plant-virus interactions is needed.
Acknowledgments
We thank Amita Rai, Theodore Moll, Miriam Khalil, Maxwell Braud, Jackson Gehan, Collin Luebbert, Jennifer Winch, Jacquelyn Leise, Stephanie Lamb, Claire Albin and Mary Lyon at the Donald Danforth Plant Science Center for their technical assistance.