Tissue-specific localization of pea root infection by Nectria haematococca. Mechanisms and consequences

Abstract

Root infection in susceptible host species is initiated predominantly in the zone of elongation, whereas the remainder of the root is resistant. Nectria haematococca infection of pea (Pisum sativum) was used as a model to explore possible mechanisms influencing the localization of root infection. The failure to infect the root tip was not due to a failure to induce spore germination at this site, suppression of pathogenicity genes in the fungus, or increased expression of plant defense genes. Instead, exudates from the root tip induce rapid spore germination by a pathway that is independent of nutrient-induced germination. Subsequently, a factor produced during fungal infection and death of border cells at the root apex appears to selectively suppress fungal growth and prevent sporulation. Host-specific mantle formation in response to border cells appears to represent a previously unrecognized form of host-parasite relationship common to diverse species. The dynamics of signal exchange leading to mantle development may play a key role in fostering plant health, by protecting root meristems from pathogenic invasion.

Figure 1.

Localized infection by N. haematococca in the (A) apical and root cap meristem (visualized with trypan blue, which stains dead cells), (B) maturation zone, (C) elongation zone/FIZ, (D) border cells, and (E) root cap. F, Dispersal of border cell mantles along the length of an inoculated root as it grows. Arrows (and inset) indicate clumps of border cells supporting fungal growth. G to I, Cross section of root apex (apical meristem, root cap meristem, root cap body) after removal of border cells from uninoculated control seedlings (G), after removal of border cell mantles from inoculated root with infection into peripheral root cap (H), and after removal of border cell mantles from a root tip infected throughout the root cap and apical meristem (I). Arrows indicate individual fungal hyphae.

Figure 2.

Germination of N. haematococca spores in situ, in response to root cap and border cell exudates. After immersion of root tips into a suspension of spores at 10⁵/mL, spores start to germinate within 1 h (arrows). Inset, Left, At 10⁶ spores/mL, chlamydospore formation occurs. Inset, Right, Fungal hyphae grow around border cells without apparent penetration.

Figure 3.

Expression of PEP genes during early stages of mantle development. A, Expression of PDA-GUS in germinating spores exposed to pisatin; B, expression of PDA-GUS in border cell mantles 24 h post inoculation; C, expression of PDA-GUS in older mantle surrounding infected root tip; and D, expression of PEP5 (left) and PEP2 (right) genes of N. haematococca at time 0 and at 24 h after inoculation. Two-day-old seedlings containing a full set of border cells were inoculated with 10⁶ spores of N. haematococca, and RNA from root tips was recovered at specific time points after inoculation, as described in “Materials and Methods.” A 100-bp ladder was used to establish agreement between expected size and actual size of fragments obtained by amplification of each gene.

Figure 4.

Inverse relationship between expression of defense genes and cell cycle genes in root tips treated with SA at a level that inhibits root growth. Relative levels of (A) chitinase mRNA and (B) cyclin mRNA were normalized to actin as a control. Seedlings were grown in cellophane pouches in which SA was added at a concentration that resulted in approximately 50% inhibition of root growth; root lengths were measured nondestructively as by Bauer (1981) before harvesting root tips for RNA-blot analysis, as by Gunawardena and Hawes (2002).

Figure 5.

Differential growth of N. haematococca in root exudates (R.E.) compared with border cells. Spores were added to samples of border cells or bulk root cap exudate, and growth was monitored over the course of 3 d, as described in “Materials and Methods.” Values represent means from four independent experiments, and ses were less than 10% of the mean.

Figure 6.

Inhibition of growth of N. haematococca in root exudates of inoculated seedlings. A, Spores (10⁵/mL) of N. haematococca 34-18 were added to samples of root exudate collected from control or inoculated seedlings, and growth was monitored over the course of 3 d, as described in “Materials and Methods.” The difference in the growth was statistically significant for the growth in the second (P value 0.009) and the third day (P value 0.006) but not for the first day (P value 0.745). B, Growth of N. haematococca 94-2-4 in exudate from seedlings inoculated with strain 34-18 and collected as described in “Materials and Methods.” Values for exudate from control or inoculated seedlings at day 2 and day 3 were statistically distinct at P < 0.05. C, Growth of M. pinoides in exudate from seedlings inoculated with N. haematococca strain 34-18 and collected as described in “Materials and Methods.” Values were not statistically distinct at P < 0.05.