Heterologous Expression of CLIBASIA_03915/CLIBASIA_04250 by Tobacco Mosaic Virus Resulted in Phloem Necrosis in the Senescent Leaves of Nicotiana benthamiana

Huanglongbing (HLB), also known as citrus greening, is the most notorious citrus disease worldwide. Candidatus Liberibacter asiaticus (CaLas) is a phloem-restricted bacterium associated with HLB. Because there is no mutant library available, the pathogenesis of CaLas is obscure. In this study, we employed tobacco mosaic virus (TMV) to express two mature secretion proteins CLIBASIA_03915 (m03915) and CLIBASIA_04250 (m04250) in Nicotiana benthamiana (N. benthamiana). Phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the two low molecular weight proteins, while no phloem necrosis was observed in the plants that expressed the control, green fluorescent protein (GFP). Additionally, no phloem necrosis was observed in the senescent leaves of N. benthamiana that expressed the null mutation of m03915 and frameshifting m04250. The subcellular localizations of m03915 and m04250 were determined by fusion with GFP using confocal microscopy. The subcellular localization of m03915 was found to be as free GFP without a nuclear localization sequence (NLS). However, m04250 did have an NLS. Yeast two-hybrid (Y2H) was carried out to probe the citrus proteins interacting with m03915 and m04250. Six citrus proteins were found to interact with m03915. The identified proteins were involved in the metabolism of compounds, transcription, response to abiotic stress, ubiquitin-mediated protein degradation, etc. The prey of m04250 was involved in the processing of specific pre-mRNAs. Identification of new virulence factors of CaLas will give insight into the pathogenesis of CaLas, and therefore, it will eventually help develop the HLB-resistant citrus.

Systemic Hypersensitive Resistance to Turnip mosaic virus in Brassica juncea is Associated With Multiple Defense Responses, Especially Phloem Necrosis and Xylem Occlusion

Systemic hypersensitive resistance (SHR) caused by Turnip mosaic virus (TuMV) was studied by light microscopy and histochemical analysis in stem cross sections of Brassica juncea (Indian mustard) plants. Ten TuMV isolates were inoculated to leaves of susceptible line JM 06006, cv. Oasis CI, which carries TuMV systemic hypersensitivity gene TuRBJU 01, and F3 progeny plants obtained from a cross between them. Systemic mosaic (SM) symptoms were induced by all 10 isolates in plants of JM 06006, and by resistance-breaking isolate NSW-3 in all cv. Oasis CI and F3 plants. With the other nine isolates, cv. Oasis CI plants developed SHR while F3 progeny plants segregated for both phenotypes; mock-inoculated control plants never became infected. Presence of SHR did not delay systemic invasion as this commenced within 2 hours after inoculation (hai) and was almost complete by 72 hai regardless of whether plants subsequently developed SHR or SM. When stem cross sections sampled 9 to 12 days after inoculation were examined for the plant defense responses, phloem necrosis, hydrogen peroxide accumulation, and additional lignin deposition, sections from plants with SHR demonstrated all of these characteristics, but sections from plants with SM or mock-inoculation did not. Based on consolidated data from all isolates except NSW-3, stems developing SHR had significantly more occluded xylem vessels (P < 0.001) compared with stems from plants developing SM or mock-inoculated plants. Both light microscopy and histochemical tests with phloroglucinol-HCl and toluidine blue O indicated that the xylem occlusions could be gels. Thus, phloem necrosis, xylem occlusion, lignification, and hydrogen peroxide accumulation were all associated with the SHR in B. juncea plants carrying TuMV hypersensitivity gene TuRBJU 01. In addition, virus inclusion bodies were fewer in sections from plants with SHR. Phloem necrosis was apparently acting as the primary cause of SHR and xylem occlusion as an important secondary cause.

Phloem Production in Huanglongbing-affected Citrus Trees

Citrus greening disease [Huanglongbing (HLB)] is the most significant and widespread threat to the citrus industry in recent history. A bacterium [Candidatus Liberibacter asiaticus (CLas)] vectored by the Asian citrus psyllid is the presumed causal agent of the disease, which results in the collapse of phloem tissue, leading to decreased productivity, chlorotic leaves, and bitter, misshapen fruit. Once infected, trees never fully recover and there currently is no cure, although foliar nutrient sprays and intensive irrigation appear to slow tree decline in some situations. Despite phloem necrosis in older tissue, new vegetative and reproductive growth occurs. Our current understanding of phloem collapse in citrus resulting from HLB is based on anatomical reports of trees in different stages of decline and does not explain the persistence of growth. Here, we present data that show new phloem cells are produced during the periodic flushes of vegetative growth and their subsequent collapse and plugging over a 6-month period. Cellular activity within the cambium and the ray parenchyma was diminished in HLB-affected petioles, suggesting an important link in the carbohydrate transport pathway is missing. Because of the short window of time during which the phloem appears healthy, the weeks immediately before and after the spring and summer flush are of critical importance for the management of citrus health.

Histo- and cytopathology of trunk phloem necrosis, a form of rubber tree (Hevea brasiliensis Müll. Arg.) tapping panel dryness

Trunk phloem necrosis (TPN) is a physiological disease of rubber tree (Hevea brasiliensis Müll. Arg.) discovered in the 1980s. It has been distinguished from rubber tree tapping panel dryness (TPD) by its macroscopic symptoms and presumed origin. But little attention has been paid to its microscopic features, and there is now some evidence that both syndromes could be linked to an impaired cyanide metabolism. In order to characterise TPN and compare it with TPD microscopically, the inner phloem of tapping panels was investigated by light and transmission electron microscopy in healthy trees and TPN-affected trees. TPN-affected phloem presented numerous and varied structural and ultrastructural features. There were signs of cellular deterioration in a great number of specialised cells, i.e. laticifers and sieve tubes, and not very specialised cells, i.e. parenchyma cells and companion cells. There were also signs of cellular dedifferentiation in other parenchymatous cells, e.g. in tylosoids and hyperplasic cells. These cells were derived from parenchyma cells that ensheath laticifers in which the latex coagulated. Numerous structural features of TPN are common to TPD, notably tylosoids associated with in situ coagulated latex, which are also known to be early structural markers of TPD and cyanide-induced. It is therefore concluded that TPN is identical to or a variant of TPD, and is a degenerative disease of rubber tree trunk phloem resembling plant stress response, programmed cell death and plant tumourigenesis in some aspects.

Real-Time PCR-Based Monitoring of DNA Pools in the Tri-Trophic Interaction Between Norway Spruce, the Rust Thekopsora areolata, and an Opportunistic Ascomycetous Phomopsis sp.

The difficulty in subculturing biotrophic fungi complicates etiological studies related to the associated plant diseases. By employing internal transcribed spacer rDNA-targeted quantitative real-time polymerase chain reaction, we now show that the heteroecious rust Thekopsora areolata, commonly associated in natural conditions to sapling shoots and cones of Norway spruce and leaves of wild bird cherry, frequently infects nursery-grown seedlings of the conifer. A spatial sampling scheme was used to investigate seedlings and saplings of Norway spruce showing phloem necrosis: the highest concentration of DNA of T. areolata was recorded in the area with necrotic phloem. The separate analysis of bark and wood tissues suggested that the initial spread of the rust to healthy tissues neighboring the infection site takes place in the bark. A Phomopsis species found to coexist with T. areolata in several seedlings showed very high DNA levels in the upper part of the lesion, and even in the visually healthy proximal tissues above the lesions, which indicates that the ascomycete, most probably a secondary invader following primary infection by T. areolata, has a latent stage during early host colonization. We hypothesize that this hemibiotrophic mode of infection contributes to the successful coexistence of Phomopsis with a biotrophic rust.