| Huanglongbing (HLB, or citrus greening) is a highly destructive citrus disease, threatening the citrus industry worldwide. The presumable causal agent of HLB is a gram-negative, phloem-inhabitingα-Proteobacteria, and includes three species, Candidatus Liberibacter asiaticus, Ca. L. africanus and Ca. L. americanus. Ca. Liberibacter is transmitted by grafting and two types of phloem-feeding psyllids, Diaphorina citri and Trioza erytreae.Ca. Liberibacter has not yet been cultured in vitro and Koch's postulates have not been fulfilled, resulting in the limited progress of associated research on HLB (e.g. the pathogenicity of Ca. Liberibacter). Furthermore, all known citrus species, varieties and citrus relatives can be affected by HLB. Currently, there is no cure for infected trees. Some citrus varieties, such as sweet orange and grapefruit, are highly sensitive to HLB, and decline in several years after infection. Only a few lemon varieties and citrus relatives have been reported to show some tolerance to HLB. Unfortunately, no resistant variety has been found yet. Control of HLB depends on use of disease-free propagating material, removal of infected trees as soon as they are detected, and attempts to control the psyllid vectors with insecticides.The infection of pathogen could result in changes of gene expression, protein profile, metabolite profile and ultrastructure of plant hosts. Studying the host responses at molecular, cellular, anatomical and physiological levels would help understand the disease mechanisms of HLB and may contribute to developing new tools to manage this devastating disease. In this study, biochemical, proteomic, microarray and microscopy approaches were applied to decipher the response of citrus to HLB bacteria. Moreover, differential responses of tolerant and susceptible citrus species were extensively compared at molecular and anatomical levels. Results and conclusions are summarized below.①Starch levels in HLB-infected sweet orange leaves with and without symptoms increased compared to healthy controls. The expression profiles of starch breakdown genes suggested that the transcription of DPE2 and MEX1 was down-regulated. In symptomless leaves, sucrose and fructose accumulated significantly in both midribs and lobes, and glucose only in the midribs; whereas maltose levels were reduced in both midribs and lobes. In leaves with symptoms, sucrose and glucose remained at high levels compared to healthy leaves, whilst no accumulation of fructose was observed; by contrast, the maltose content decreased. It is indicated that the carbohydrate metabolism is imbalanced and starch breakdown is inhibited in HLB-diseased sweet orange. Additionally, the cell-wall-bound invertase activity was induced in both types of leaves on diseased plants. Collectively, it is suggested that the life style of Ca. L. asiaticus is biotrophic.②Differetially expressed proteins in HLB-diseased sweet orange were indentified by proteomic approach (iTRAQ). Twenty and 10 proteins were differentially expressed in HLB-diseased leaves with and without symptoms, respectively, compared with mock-inoculated plants.③Four miraculin-like proteins (MLP), chinase, Cu/Zn superoxide dismutase and lipoxygenase were highly induced by 3.6-13.7 folds upon HLB infection. These proteins are mainly involved in stress/defense response, suggesting the host defense system is activated by HLB bacteria; and they may also be developed as biomarkers for HLB diagnosis.④Comparative transcriptional profiling showed that 684, 411 and 1053 probe sets differentially expressed at 5, 17 and 27 weeks after inoculation (WAI) of Ca. L. asiaticus in rough lemon data, and 391, 433 and 366 probe sets in sweet orange data. A wide range of pathways, such as photosynthesis, cell wall, transcription factors, hormone metabolism, stress response and carbohydrate metabolism, were significantly affected in diseased rough lemon and sweet orange.⑤Similar changes were found in both data sets, e.g. repression of photosynthesis-related genes and a set of MYB-related transcription factor genes. However, a number of pathways were differentially deregulated not only among the three time points within species but also between the two species. For example, ethylene signaling pathway was significantly deregulated in diseased sweet orange; on the contrary, more changes of auxin, brassinosteroid and salicylic acid pathway genes were observed in rough lemon data. Cell wall associated processes were significantly down-regulated at 5 WAI (early stage) but up-regulated at 27 WAI (late stage) in diseased rough lemon; less changes were observed in sweet orange data. In addition, the expression change pp2 involved in HLB-associated phloem plugging was up-regulated in diseased sweet orange, but not in rough lemon. These data may provide candidate genes for improving citrus tolerance to HLB disease by genetic engineering. The mechanisms of HLB tolerance in rough lemon is discussed. ⑥Microscopy analysis showed that phloem collapse, plugged sieve elements and accumulation of starch were observed in symptomatic leaves of both diseased rough lemon and sweet orange. In symptomless leaves, less changes were found in rough lemon than in sweet orange. Futhermore, the degradation of phloem fibers and starch depletion were found in HLB-diseased sweet orange roots, but not in rough lemon roots. Taken together, rough lemon is less affected by HLB than sweet orange at anatomical level, providing further evidence of tolerance in rough lemon and susceptibility of sweet orange.⑦Phloem transport experiment showed that mature leaves of HLB-diseased rough lemon transported carboxyfluorescein as normal or nearly normal as healthy controls. By contrast, phloem transportation of HLB-diseased sweet orange was obviously inhibited. Thus, the sink organs of infected rough lemon, especially the roots, can still get abundant photoassimilates to maintain growth; while diseased sweet orange is likely to have stunt growth or even death, due to the lack of sugars transporting to the roots.
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