Epifluorescence microscopy imaging of phytoplasmas in embedded leaf tissues using DAPI and SYTO13 fluorochromes

The use of DNA-specific dyes, i. e. DAPI, is extensively reported for phytoplasma detection in fresh plant materials. However, fluorescence-based microscopy and imaging of fresh tissues often evidences technical limitations which are more significant in infected tissues, because phenolic and other defense-related compounds accumulate in the cell wall and in the vacuole making difficult sample preparation. In this paper we describe a method based on the use of epifluorescence microscopy and the DNA probes DAPI and SYTO13\uae for phytoplasma visualization in resin-embedded plant tissues. The method allows detection of phytoplasmas and it is recommended for tissues that are recalcitrant to conventional imaging

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani,’ the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Western blot analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

Phytoplasma infection in tomato is associated with re-organization of plasma membrane, ER stacks, and actin filaments in sieve elements

Phytoplasmas, biotrophic wall-less prokaryotes, only reside in sieve elements of their host plants. The essentials of the intimate interaction between phytoplasmas and their hosts are poorly understood, which calls for research on potential ultrastructural modifications. We investigated modifications of the sieve-element ultrastructure induced in tomato plants by ‘Candidatus Phytoplasma solani’, the pathogen associated with the stolbur disease. Phytoplasma infection induces a drastic re-organization of sieve-element substructures including changes in plasma membrane surface and distortion of the sieve-element reticulum. Observations of healthy and stolbur-diseased plants provided evidence for the emergence of structural links between sieve-element plasma membrane and phytoplasmas. One-sided actin aggregates on the phytoplasma surface also inferred a connection between phytoplasma and sieve-element cytoskeleton. Actin filaments displaced from the sieve-element mictoplasm to the surface of the phytoplasmas in infected sieve elements. Expression analysis revealed a decrease of actin and an increase of ER-resident chaperone luminal binding protein (BiP) in midribs of phytoplasma-infected plants. Collectively, the studies provided novel insights into ultrastructural responses of host sieve elements to phloem-restricted prokaryotes

Reactions and change of forisome position in <i>Vicia faba</i> (A-E) and forisome location in <i>Phaseolus vulgaris</i> sieve elements (F-O) in response to remote heating (forisomes are marked by asterisks).

<p>A. <i>Vicia faba</i> forisome (upstream position 1). B-E <i>Vicia faba</i> forisome (downstream position, position1). B. Initial position at an angle of about 10° to the longitudinal axis, C. Dispersion in response to a remote heat shock, D. Re-condensation, 4 min after the stimulus, position parallel to the longitudinal axis, E. 5 min after stimulus, position at an angle of about 10° to the longitudinal axis. F. <i>Phaseolus vulgaris</i> forisome (upstream position). G-I Long-distance ovement of a non-primed condensed Phaseolus vulgaris forisome from the downstream position 1 to the upstream position 4, J-L Short-distance movement of a non-primed condensed <i>Phaseolus vulgaris</i> forisome from the downstream position 1 (J.), via the central position 4 (K) back to the original downstream position 1 (L). M-O No position change of a dispersed <i>Phaseolus vulgaris</i> forisome. Direction of flow in G to O from right to left.</p

Forisome reactivity in response to remote burning of intact non-primed <i>Phaseolus vulgaris</i> plants as related to its location.

<p>“Reaction” includes both dispersion or movement.</p

A-C Dispersion and re-condensation of a forisome in a LatA pretreated non-primed <i>Phaseolus vulgaris</i>plant in response to a remote heat stimulus. D. Relationship between dispersion and re-condensation times.

<p>The stippled line indicates the relationship between dispersion and re-condensation time.</p

A. Average acropetal and subsequent basipetal movement velocities of originally downstream located condensed <i>Phaseolus vulgaris</i> forisomes after a distant heat stimulus in non-primed plants. B. Response lag times of location changes of forisomes in <i>Phaseolus</i> vulgaris and the distances of movement.

<p>The stippled line indicates the relationship between dispersion and re-condensation time.</p

Forisome positions at diverse sieve-element locations in <i>Vicia faba</i> (A-D) and <i>Phaseolus vulgaris</i> (E-H).

<p>A. and E. basal (downstream); B. and F. central; C. and G. apical (upstream). D. and H. Percentage of forisomes at each location and position. The black areas indicate forisomes in contact with the sieve-element side facing the companion cell. Different letters indicate significant differences between the positions of forisomes.</p

Reactivity of basal forisomes pretreated with LatA in response to remote burning of intact non-primed <i>Phaseolus vulgaris</i> plants as related to its position, without prior priming.

<p>“Reaction” includes both dispersion and movement.</p