Dynamics of Candidatus Liberibacter asiaticus Movement and Sieve-Pore Plugging in Citrus Sink Cells

Abstract

Citrus greening or Huanglongbing (HLB) is caused by the phloem-limited intracellular Gram-negative bacterium Candidatus Liberibacter asiaticus (CLas). HLB-infected citrus phloem cells undergo structural modifications that include cell wall thickening, callose and phloem protein induction, and cellular plugging. However, very little is known about the intracellular mechanisms that take place during CLas cell-to-cell movement. Here, we show that CLas movement through phloem pores of sweet orange (Citrus sinensis) and grapefruit (Citrus paradisi) is carried out by the elongated form of the bacteria. The round form of CLas is too large to move, but can change its morphology to enable its movement. CLas cells adhere to the plasma membrane of the phloem cells specifically adjacent to the sieve pores. Remarkably, CLas was present in both mature sieve element cells and nucleated nonsieve element cells. The sieve plate plugging structures of host plants were shown to have different composition in different citrus tissues. Callose deposition was the main plugging mechanism in the HLB-infected flush, where it reduced the open space of the pores. In the roots, pores were surrounded by dark extracellular material, with very little accumulation of callose. The expression of CALLOSE SYNTHASE7 and PHLOEM PROTEIN2 genes was upregulated in the shoots, but downregulated in root tissues. In seed coats, no phloem occlusion was observed, and CLas accumulated to high levels. Our results provide insight into the cellular mechanisms of Gram-negative bacterial cell-to-cell movement in plant phloem.

Figure 1.

Phloem pore plugging in HLB-infected flush. A to F, TEM micrographs of sieve plates in Madam Vinous sweet orange flush. Arrows indicate open pores, whereas arrowheads indicate a blocked pore. A, High-magnification image showing sieve pore structure. Arrows are pointing at two pores. Callose(Ca) is the lighter-color material present next to the cell wall (CW) along the pores. B to D, Cross section of sieve plates (SP). B, Open pores in uninfected plants. Pores contain callose, but an open pathway is clear (arrow). C, Mixed situation in an infected asymptomatic plant. Some pores are open (arrows) whereas others are occluded (arrowhead). D, Completely sealed pores in infected symptomatic plant (arrowheads). E and F, Longitudinal sections of sieve plates, showing the accumulation of either low levels (E) or high levels (F) of callose. G, Average opening size of sieve plate pores in uninfected, infected asymptomatic, and infected symptomatic sweet orange flush. Different letters indicate statistically significant difference (P < 0.0001; Tukey’s honestly significant difference). Error bars are se, N (uninfected) = 77, N (infected asymptomatic) = 159 and N (infected symptomatic) = 119. SE, sieve elements.

Figure 2.

Phloem plugging in HLB-infected roots. A to K, TEM images of sieve area in healthy (A, B, E, and F) and HLB-infected (C, D, G, and K) roots of Duncan grapefruit (A–D), Valencia sweet orange (E–H), and Madame Vinous sweet orange (I–K). A, B, E, and F, Sieve pores (SP) of healthy grapefruit and sweet orange containing callose (Ca) next to the darker cell wall (CW) along the pores. C, D, G, and H, Sieve pores of grapefruit and sweet orange HLB-infected plants mainly contain extracellular deposits of a dark material (DM). These deposits are formed between the plasma membrane (pm) and the pore cell wall. Sieve elements also contain starch granules (SG). CLas cells are marked with an asterisk (*). I to K, Deposited dark material together with thin collars of callose in sieve pores from infected sweet orange.

Figure 3.

CalS and PP2 gene expression analyses. A, RQ of CalS7 and PP2 transcripts normalized with GAPDH and ActB reference genes, in different tissues of noninfected and HLB-infected Citrus macrophylla plants. Comparisons with asterisk (*) indicates significant difference (P < 0.05; multiple Student’s t test). B, CalS and PP2 gene expression in lateral veins of healthy and CLas-infected (blotchy mottled) Duncan grapefruit leaves. Transcripts normalized with ActB reference gene. Error bars are se, n = 3.

Figure 4.

Phloem pores in HLB-infected seed coats. A to C, TEM images of phloem and sieve plates in HLB-infected sweet orange seed coats. A, Phloem sieve elements (se) are filled with CLas bacteria with open sieve plates (SP) pores. B, Higher magnification of the sieve plate. CLas bacterial cells (marked with asterisks [*]), including dividing bacteria (marked by arrow), are present, and pores are open. C, Seed coat phloem. Very high numbers of bacterial cells are found, even inside nucleated cells (bacteria marked with asterisks). In addition, some cells are full of deteriorating bacteria (DB). Nu, nucleus.

Figure 5.

CLas movement between phloem cells TEM cross-section images of HLB-infected sweet orange seed coats. A to C, Elongated CLas bacteria passing through the phloem pores. Arrows point to the crossing bacteria. D to F, Movement of the round form of the bacteria between cells. Arrows point to the bacteria changing between the elongated and circular forms.

Figure 6.

CLas adhesion to host plasma membrane observed in TEM cross-section images of HLB-infected Duncan grapefruit (A, B, and F) and sweet orange (C–E) seed coats (A and B), flush (C and D), and roots (E and F). A to C, Attachment of CLas bacteria to the host cell membrane adjacent to the phloem pores through an unknown filamentous material (arrows). D to F, Attachment of CLas bacteria to the host cell membrane adjacent to the phloem pores through an anchor-like link (arrows).