Interspecific RNA interference of SHOOT MERISTEMLESS-like disrupts Cuscuta pentagona plant parasitism

Abstract

Infection of crop species by parasitic plants is a major agricultural hindrance resulting in substantial crop losses worldwide. Parasitic plants establish vascular connections with the host plant via structures termed haustoria, which allow acquisition of water and nutrients, often to the detriment of the infected host. Despite the agricultural impact of parasitic plants, the molecular and developmental processes by which host/parasitic interactions are established are not well understood. Here, we examine the development and subsequent establishment of haustorial connections by the parasite dodder (Cuscuta pentagona) on tobacco (Nicotiana tabacum) plants. Formation of haustoria in dodder is accompanied by upregulation of dodder KNOTTED-like homeobox transcription factors, including SHOOT MERISTEMLESS-like (STM). We demonstrate interspecific silencing of a STM gene in dodder driven by a vascular-specific promoter in transgenic host plants and find that this silencing disrupts dodder growth. The reduced efficacy of dodder infection on STM RNA interference transgenics results from defects in haustorial connection, development, and establishment. Identification of transgene-specific small RNAs in the parasite, coupled with reduced parasite fecundity and increased growth of the infected host, demonstrates the efficacy of interspecific small RNA-mediated silencing of parasite genes. This technology has the potential to be an effective method of biological control of plant parasite infection.

Figure 1.

Parasitism by Dodder. (A) Tobacco infected with dodder. (B) Dodder strand showing embedded haustoria (white arrowheads) and prehaustoria (black arrowhead). (C) Longitudinal section of a dodder strand with vascular tissue (V) and initiating haustoria (black arrows). (D) Longitudinal section of a dodder strand in high magnification showing developing haustoria. (E) Dodder haustoria emerging from the stem epidermis; the asterisk marks file cells, and the arrowhead marks digitate cells in the prehaustorium. (F) Transverse section of a tobacco stem with an attached dodder strand. Mature haustoria (H) of dodder (marked with a yellow line) are embedded in the tobacco stem (demarcated with a green line) and Xylic searching hyphae (arrow) have contacted the host xylem (Xy), while Phloic hyphae (asterisks) contact the host phloem (Ph). (G) Transverse section of a tobacco stem (to) with attached dodder parasite (p) stained with Aniline Blue. Mature haustorium shows a central column of Xylic hyphae (arrows) that have contacted the host xylem, and peripheral to that, Phloic hyphae (marked with asterisks) that contact the host phloem. in, host-parasite interface. (H) Transverse section of a tobacco stem with attached dodder parasite showing a haustorium sending out numerous searching hyphae (arrows) toward the host vascular tissue. Bars = 100 µm.

Figure 2.

Expression of STM during Haustorial Development. (A) qRT-PCR showing expression of STM in dodder apex and stem strands with haustoria (a, apex; h, haustorium; p, prehaustorium; sh, strand haustorium; sph, strand prehaustorium). Error bars represent sd from the mean. (B) Dodder stem showing STM expression in vascular and adjacent tissue. (C) Sense control. (D) and (E) Prehaustorium antisense and sense. Prior to emergence from stem where (D) is showing expression in digitate cells and file cells compared with sense control (E). (F) and (G) Developing haustoria. STM expression is seen in developing vascular strand (F) compared with sense control (G). Bars = 100 µm.

Figure 3.

STM Downregulation in Dodder. (A) qRT-PCR showing reduced dodder STM expression in both attached and independent RNAi transgenic lines SUC2-3 and SUC2-7. (B) Distribution of the reads across the STM gene fragment in the region targeted for RNAi and reads outside the targeted region. Raw read counts are shown here to visualize the distribution.

Figure 4.

Coverage of Small RNA Reads across the Hairpin Construct in Dodder. The read counts (peaks) have been overlayed on top of a graphic of the hairpin construct that has the SUC2 promoter and STM and Knat1-3b in forward and reverse orientation surrounding a maize waxy intron and the Nos terminator. The inset shows the starting, ending, and size of the various regions in the construct. Note that some reads are detected in the polylinker region as well as the intron.

Figure 5.

Dodder Grown on Transgenic Hosts Exhibits Reduced Vigor. (A) Dodder grows vigorously on wild-type tobacco compared with SUC2 RNAi transgenic host. WT, wild type. (B) Premature transition to flowering is seen in the dodder growing on transgenics. [See online article for color version of this figure.]

Figure 6.

Normalized Read Counts of Small RNA Molecules. Reads mapping to the microRNAs that regulate flowering, at-miR172c and d, and phase change, at-miR159a. WT, wild type.

Figure 7.

RNAi of STM Alters Haustorial Growth in Dodder. Searching hyphae (colored light green) development on untransformed (A) and transgenic tobacco ([B] and [C]). The host is demarcated with a green line and the parasite with a yellow line. Host xylem is labeled Xy, and H is haustoria. Bars = 100 µm.