The growth of grass leaves is largely a function of cell division within the leaf primordium and cells immediately above, which then cease dividing and undergo a phase of rapid expansion. Grass cells enlarge within the leaf expansion zone, a region that encompasses the basal few centimeters of the emerging leaf (Snyder et al., 1990). Plant cells within this zone are expanding to a greater or lesser extent while being displaced away from the leaf base by the continued production of cells beneath. The leaf effectively grows at the base by intercalary extension. Endophyte hyphae present in the leaf expansion zone appear to be attached to host cell walls (Christensen et al., 2008). In addition, the hyphae may have lateral branches (Fig. 14-25), some of which connect longitudinal hyphae on either side of files of expanding plant cells. Given that fungi are understood to grow exclusively by apical tip extension, how hyphal filaments and lateral branches remain intact when attached to enlarging leaf cells was unclear until recently (Christensen et al., 2008; Christensen and Voisey, 2007). Hyphae in the leaf expansion zone extend, not by apical growth, but by intercalary growth along the length of the filament (Christensen et al., 2008). Cumulative increases in the intercalary length of hyphae enable the endophyte to grow in the expansion zone in a manner analogous to the host. This adaptation enables endophytes transmitted by seed to circumvent physical stretching forces during leaf expansion that would otherwise cause hyphal fragmentation.
Wall building away from the hyphal tip, an essential requirement of intercalary hyphal extension, is a continuous process, as shown by the increase in wall thickness as hyphae age (Christensen et al., 2002).
The following evidence supports the hypothesis that Epichloë and Neotyphodium hyphae grow by intercalary extension both in growing leaves and in the internodal regions of developing reproductive tillers, and possibly in other parts of the plants where plant cells increase in size.
1. Lateral branches and the hyphae on which they have formed remain intact within elongating leaves (Christensen et al., 2008).
2. The distance between branches on GFP-expressing hyphae in the leaf expansion zone of elongating leaves increases with time, as measured using time-lapse photography (Christensen et al., 2008).
3. Hyphal ultrastructure in elongating leaves is related to the distance of the compartments from the leaf base (the age of leaf tissue), rather than the distance from the hyphal tip, suggesting that all hyphae in the same region of the leaf are at a similar stage of development (Christensen et al., 2008). Evidence that hyphae colonize each section of leaf at a similar time is consistent with intercalary hyphal growth driven by their attachment to upwardly expanding leaf tissues.
Fig. 14-25. Branched hyphae of a GFP-expressing strain of Epichloë festucae within the leaf expansion zone of a perennial ryegrass leaf. The two dark longitudinal bands indicate the position of vascular bundles.
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