Investigation of host cell wall degrading strategies of Pseudomonas syringae pv. actinidiae (Psa)
Kiwifruit (Actinidia deliciosa and Actinidia chinensis) is cultivated in several countries including Italy, France, Chile, China, Japan, Korea and New Zealand and is an important income earner for a majority of these countries. In New Zealand, kiwifruit earns approximately USD 714 million per annum. In the recent past, the New Zealand kiwifruit industry was threatened by an outbreak of bacterial canker caused by Pseudomonas syringae pv. actinidiae (Psa). The invasion strategies and the mobility of Psa pathogen in the kiwifruit plant have not been fully described. Recent comparative genomic studies of Psa strains and other P. syringae pathovars that also infect woody hosts indicate similarities in genetic makeup among these pathovars. Interestingly, these pathovars contain genes capable of producing cell wall degrading enzymes, thus suggesting that Psa possesses the same capability. However, no previous research demonstrated that any P. syringae pathovar degrades the host’s cell walls or any cell wall component. This research investigated whether Psa produces xylanases to degrade cell wall material of kiwifruit stems and facilitate systemic mobility of the pathogen from the infection site. The research also investigates whether vessel anatomy and xylem architecture play a role in the systemic mobility of the pathogen within the plant. Chapter Two of the thesis determines whether Psa is able to multiply and grow on kiwifruit wood-containing medium and whether Psa synthesises any xylanase to degrade cell wall material contained in the kiwifruit wood. For this purpose, the genome of Psa was analysed to ascertain whether sequences homologous to well-known cell wall degrading enzymes were present in the genome. The degree of similarity of the identified genes was compared to the cell wall degrading genes of other Pseudomonas woody host pathogens. Six genes encoding for well-known cell wall degrading enzymes were identified, with the degree of amino acid similarity varying between 58% and 98%. Patterns of Psa growth were examined in three media: standard nutrient broth, xylan and casein media and minimal media supplemented with kiwifruit cell wall material, called kiwifruit xylem medium. Growth of the bacterium was observed in the kiwifruit xylem medium, however, the increase in CFU (Colony Forming Units) counts in kiwifruit xylem medium was low compared to that in the other two media. A characteristic bacterial growth pattern was observed in the standard nutrient broth media. In the kiwifruit xylem media, bacteria counts declined initially, then briefly recovered. In vitro enzyme assays were conducted for Psa in the kiwifruit xylem medium to determine the activity of xylanase enzyme. Two types of assays were conducted: 3,5-Dinitrosalicylic acid (DNSA) and Remazol Brilliant Blue (RBB). A significant xylanase activity was detected only in the Psa cultured in minimal medium supplemented with 0.5% kiwifruit xylem using the DNSA assay. However, no xylanase activity was detected using the RBB. It was concluded that Psa is capable of producing an active xylanase enzyme, and that synthesis of the enzyme is induced under low nutrient conditions and in the presence of kiwifruit xylem cell wall material. Chapter Three describes investigations of xylanase activity in planta. Twenty mature Actinidia chinensis Planch. var. chinensis ‘Hort16A’ plants were used. Ten plants were left un-inoculated and ten plants were inoculated with Psa. Disease symptoms appeared in the inoculated plants after which both inoculated and non-inoculated shoots were harvested. Psa was re-isolated from infected plants and duplex PCRs were conducted to confirm that symptoms were due to Psa infection. RBB and DNSA assays were conducted on ground kiwifruit stem pieces to ascertain putative xylanase activity. The RBB assay indicated xylanase activity in inoculated kiwifruit stem pieces. RBB assay parameters were varied to ascertain whether the xylanase activity in infected tissues was consistent with enzymatic activity. Strength tests were conducted on infected and non-infected kiwifruit shoots to determine whether infection affected stem structural integrity. The average strength per mm thickness of non-infected kiwifruit xylem was significantly higher than that of infected xylem. RNA was extracted from both inoculated and non-inoculated stems and PCR conducted with primers specific to the xylanase genes identified in the Psa genome. One bacterial xylanase gene was expressed during infection of the kiwifruit stems. The observed reduction in stem strength was consistent with the xylanase activity detected using the RBB assay and the expression of the bacterial xylanase gene during infection. Chapter Four investigated kiwifruit vessel lengths, the frequency of open vessel connections between kiwifruit stems and leaves, and the potential for movement of Psa from leaf inoculation sites into the supporting stem. Silicone injection was used to document xylem vessel length distribution within mature one-year-old stems of three kiwifruit cultivars that differ in Psa susceptibility. There was no correlation between average vessel lengths and susceptibility. The air injection technique was used to estimate maximum vessel lengths in Hort16A seedlings and vessel connections from leaf to stem. It was observed that the maximum xylem vessel length of Hort16A kiwifruit seedlings was 345 mm, that 71% of leaves had an open vessel from the stem to the leaf blade, and that 79% of leaves had an open vessel from stem to the petiole. The average maximum vessel length and plant height were positively correlated. A negative correlation was observed between height of the plant and the number of nodes with vessels extending into leaves. Psa was observed to move in both basipetal and acropetal directions when the leaf blade of Hort16A seedlings was inoculated, and in the majority of leaves was detected in the petiole or stem when either the tip or base of the leaf lamina was inoculated. Psa movement exceeded the boundaries of known open vessel connections between stem and leaf, suggesting that if Psa was mobile in the xylem, then vessel end walls were not a significant barrier to movement. This finding supports the conclusion that Psa has the ability to overcome plant cell wall barriers to movement. The overall findings of this thesis are that Psa does actively produce at least one cell wall degrading enzyme during infection of kiwifruit tissue, and that cell wall degrading enzymes contribute to an active invasion mechanism by this pathogen. For this reason Psa movement within the plant is not limited by cell wall barriers or the architecture of the xylem vascular system.
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