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.

Plant hairy roots enable high throughput identification of antimicrobials against Candidatus Liberibacter spp.

“…Unlike PCR/Sanger sequencing or immuno-labeling approaches, TEM alone is not a confirmatory test to identify pathogens. Unfortunately, immuno-labeling is technically challenging to perform for C Lso/ C Las detection, primarily due to limitations of existing antibodies (only available for C Las) and the low-titers and highly variable nature of C Lso/ C Las accumulation in infected plant tissues 41 . Thus far, C Las immuno-labeling was primarily successful in insect-vector 42 .…”

Bacterial Vector-Borne Plant Diseases: Unanswered Questions and Future Directions

“…Accumulation in sink tissues, including roots and growing tissues, is often observed in phloem-limited pathogens; however, their distribution within the plant is variable ( Figure 1 ). This distribution extends to individual sieve tubes in the phloem where, for example, CLas massively proliferates in some tubes while barely colonizing others ( Achor et al, 2020 ; Hartung et al, 2010 ), and spiroplasmas accumulate near undifferentiated phloem cells ( Bové et al, 2003 ). Recovery of apple trees from apple proliferation phytoplasma is associated with increased callose depositions that prevent recolonization of the apple tree crowns during the spring and with enhanced overall defense responses of recovered apple trees ( Musetti et al, 2010 ).…”

“…Similar surface proteins were also found in phytoplasmas and may play an analogous role ( Kakizawa et al, 2006a , 2006b ). Liberibacters and mollicutes use their pleomorphic shape to cross the tight space between sieve plates, even when they are fortified by callose ( Figure 1 ) ( Achor et al, 2020 ) ( Waters and Hunt, 1980 ). Phytoplasmas have been shown to adhere to the inner surface of the sieve tube membranes ( Marcone, 2009 ), and liberibacters often aggregate near sieve pores ( Achor et al, 2020 ), a feature that may facilitate movement ( Figure 1 ).…”

Development of genetically modified citrus plants for the control of citrus canker and huanglongbing

“…In the phloem sieve plates callose regulates size exclusion limit of plasmodesmata connections limiting the cell-to-cell movement of molecules (Ellinger and Voigt 2014). Callose deposition reduces the open space of the pores in the sieve plate and resulted in plugging of the HLB-infected flush (Achor et al 2020).…”